CN103223177A - Treatment of lipid transport and metabolism gene related diseases by inhibition of natural antisense transcript to a lipid transport and metabolism gene - Google Patents

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of a Lipid transport and metabolism gene, in particular, by targeting natural antisense polynucleotides of a Lipid transport and metabolism gene. The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of a Lipid transport and metabolism genes.

Description

By suppression therapy lipid transfer and metabolic gene relevant disease at the natural antisense transcript of lipid transfer and metabolic gene

The application is that international filing date is that May 6, international application no in 2010 are PCT/US2010/033908, the application number that enters country's stage is 201080030706.2, denomination of invention is divided an application for the PCT application of " by suppression therapy lipid transfer and the metabolic gene relevant disease at the natural antisense transcript of lipid transfer and metabolic gene ".

Invention field

The application requires the U.S. Provisional Patent Application submitted on May 6th, 2009 number 61/175,930, the U.S. Provisional Patent Application of submitting on May 7th, 2009 number 61/176,267, the U.S. Provisional Patent Application of submitting on May 22nd, 2009 number 61/180,646, the U.S. Provisional Patent Application of submitting on October 2nd, 2009 number 61/248,212 and the U.S. Provisional Patent Application submitted on August 19th, 2009 number 61/235,227 priority, its integral body is incorporated herein by reference.

Embodiment of the present invention comprise regulates lipid transfer and the expression of metabolic gene and correlation molecule and/or the oligonucleotide of function.

Background of invention

DNA-RNA and RNA-RNA hybridization are important for many aspects of nucleic acid function, comprise dna replication dna, transcribe and translate.Hybridization also is crucial for the various technology that detect specific nucleic acid or change its expression.Antisense nucleotide is for example by destroying gene expression with target RNA hybridization, RNA interfering montage thus, transcribes, translates and duplicate.Antisense DNA has the DNA-RNA hybrid and serves as the supplementary features that are used for by the substrate of ribonuclease H digestion, and it is the activity that exists in most cell types.Antisense molecule can be delivered in the cell, and as the situation for oligodeoxynucleotide (ODNs), or they can be expressed as the RNA molecule by endogenous gene.FDA has ratified antisense drug VITRAVENE (being used for the treatment of the cytomegalovirus retinitis) in the recent period, and reflection antisense thing has treatment effectiveness.

General introduction

Provide this general introduction to present general introduction of the present invention, briefly to point out character of the present invention and purport.The understanding of explaining or limiting claim scope or implication being not used in is followed in its proposition.

In one embodiment, the invention provides the method for the effect that is used to suppress the natural antisense transcript, one or more antisense oligonucleotides in its any zone by using targeting natural antisense transcript cause should having mutually the rise of adopted gene.This paper considers that also the inhibition of natural antisense transcript can reach by siRNA, ribozyme and micromolecule, and this is regarded as within the scope of the invention.

Embodiment provide in vivo or external adjusting patient cell or tissue in lipid transfer and the method for metabolic gene polynucleotide function and/or expression, it comprises contacts the antisense oligonucleotide of 30 nucleotide of described cell or tissue and length 5 –, the reverse complemental body of wherein said oligonucleotide and polynucleotide has at least 50% sequence homogeneity, and described polynucleotide are included in the nucleotide 1 to 1299 of SEQ ID NO:8,1 to 918 of SEQ ID NO:9,1 to 1550 of SEQ ID NO:10,1 to 329 of SEQ ID NO:11,1 to 1826 of SEQ ID NO:12,1 to 536 of SEQ ID NO:13,1 to 551 of SEQ ID NO:14,1 to 672 of SEQ ID NO:15,1 to 616 of SEQ ID NO:16,1 to 471 of SEQ ID NO:17,1 to 707 of SEQ ID NO:18,1 to 741 of SEQ ID NO:19,1 to 346 of SEQ ID NO:20,1 to 867 of SEQ ID NO:21, SEQ ID NO:22 1 to 563 in 5-30 continuous nucleotides (Fig. 3); Thereby in vivo or lipid transfer and metabolic gene polynucleotide function and/or expression in the external adjusting patient cell or tissue.

In a further preferred embodiment, the natural antisense sequence of oligonucleotide targeting lipid transfer and metabolic gene polynucleotide, the nucleotide shown in the SEQ ID NO:8-22 for example, and about its any variant, allele, homologue, mutant, derivant, fragment and complementary series.The example of antisense oligonucleotide is (Fig. 4-9) shown in SEQ ID NOS:23-263.

Another embodiment provide in vivo or external adjusting patient cell or tissue in lipid transfer and the method for metabolic gene polynucleotide function and/or expression, it comprises contacts the antisense oligonucleotide of 30 nucleotide of described cell or tissue and length 5 –, and the reverse complemental body of the antisense of wherein said oligonucleotide and lipid transfer and metabolic gene polynucleotide has at least 50% sequence homogeneity; Thereby in vivo or lipid transfer and metabolic gene polynucleotide function and/or expression in the external adjusting patient cell or tissue.

Another embodiment provide in vivo or external adjusting patient cell or tissue in lipid transfer and the method for metabolic gene polynucleotide function and/or expression, it comprises contacts the antisense oligonucleotide of 30 nucleotide of described cell or tissue and length 5 –, wherein said oligonucleotide with have at least 50% sequence homogeneity at the antisense oligonucleotide of lipid transfer and metabolic gene antisense polynucleotides; Thereby in vivo or lipid transfer and metabolic gene polynucleotide function and/or expression in the external adjusting patient cell or tissue.

In a preferred embodiment, compositions comprises and bonded one or more antisense oligonucleotides of justice and/or antisense lipid transfer and metabolic gene polynucleotide is arranged.

In a further preferred embodiment, oligonucleotide comprises nucleotide one or more modifications or that replace.

In a further preferred embodiment, oligonucleotide comprises the key of one or more modifications.

In the another one embodiment, the nucleotide of modification comprises the base of the modification that comprises thiophosphate, methyl phosphonate, peptide nucleic acid(PNA), 2 '-O-methyl, fluorine or carbon, methylene or other lock nucleic acid (LNA) molecule.Preferably, the nucleotide of modification is the lock nucleic acid molecules, comprises α-L-LNA.

In a further preferred embodiment, oligonucleotide is subcutaneous, intramuscular, intravenous or intraperitoneal are applied to the patient.

In a further preferred embodiment, oligonucleotide is used in pharmaceutical composition.Therapeutic scheme comprises to the patient uses antisense compounds at least one time, yet this treatment can be revised as a plurality of dosage that comprise through after a while.Treatment can be made up with the therapy of one or more other types.

In a further preferred embodiment, oligonucleotide is encapsulated in the liposome or with carrier molecule (for example cholesterol, tat peptide) and is connected.

Other aspects are described hereinafter.

The accompanying drawing summary

Fig. 1

Figure 1A is PCR in real time result's figure, shows to use compared with the control to use siRNA oligonucleotide that Lipofectamine 2000 introduces and handle the multiple variation+standard deviation in ABCA1 mRNA behind the 518A2 cell.PCR in real time result shows, 48 h after using at a processing among the siRNAs of ABCA1 antisense AK311445 design, and the level of ABCA1 mRNA significantly increases in the 518A2 cell.The bar (Bars) that is expressed as CUR-0512, CUR-0519 corresponds respectively to the sample of handling with SEQ ID NOS:23-25.

Figure 1B is PCR in real time result's figure, shows to use compared with the control to use thiophosphate oligonucleotide that Lipofectamine 2000 introduces and handle the multiple variation+standard deviation in ABCA1 mRNA behind the 518A2 cell.PCR in real time result shows, 48 h after using at six processing in the oligomer of ABCA1 antisense AK311445 design, and the level of ABCA1 mRNA significantly increases in the 518A2 cell.The bar that is expressed as CUR-1214 to CUR-1222 corresponds respectively to the sample of handling with SEQ ID NOS:26-34.

Fig. 1 C is PCR in real time result's figure, shows to use compared with the control to use thiophosphate oligonucleotide that Lipofectamine 2000 introduces and handle the multiple variation+standard deviation in ABCA1 mRNA behind the 3T3 cell.PCR in real time result shows, 48 h after using at three processing in the oligomer of mice ABCA1 antisense BF133827 design, and the level of ABCA1 mRNA significantly increases in the 3T3 cell.The bar that is expressed as CUR-1087 to CUR-1090, CUR-1092 and CUR-1091 corresponds respectively to SEQ ID NOS:35-38,40 and 39 samples of handling.

Fig. 1 D is PCR in real time result's figure, shows to use compared with the control to use siRNA that Lipofectamine 2000 introduces and thiophosphate oligonucleotide and handle the multiple variation+standard deviation in LCAT mRNA behind the Hek293 cell.PCR in real time result shows, 48 h after using at three processing in the oligomer of LCAT antisense Hs.668679 design, and the level of LCAT mRNA significantly increases in the Hek293 cell.The bar that is expressed as CUR-0476, CUR-0478, CUR-0822, CUR-0820 and CUR-0819 corresponds respectively to SEQ ID NOS:41,42,58,56 and 55 samples of handling.

Fig. 1 E is PCR in real time result's figure, shows to use compared with the control to use siRNA oligonucleotide that Lipofectamine 2000 introduces and handle the multiple variation+standard deviation in LCAT mRNA behind the HepG2 cell.PCR in real time result shows, 48 h after using at two processing in the oligomer of LCAT antisense Hs.668679 design, and the level of LCAT mRNA significantly increases in the HepG2 cell.The bar that is expressed as CUR-0476, CUR-0478, CUR-0444, CUR-0446, CUR-0448 and CUR-0450 corresponds respectively to SEQ ID NOS:41,42 and the sample handled of 44-47.

Fig. 1 F is PCR in real time result's figure, shows to use compared with the control to use thiophosphate oligonucleotide that Lipofectamine 2000 introduces and handle the multiple variation+standard deviation in LCAT mRNA behind the HepG2 cell.PCR in real time result shows, 48 h after using at a processing in the oligomer of LCAT antisense Hs.668679 design, and the level of LCAT mRNA significantly increases in the HepG2 cell.The bar that is expressed as CUR-0819, CUR-0818, CUR-0817, CUR-0816, CUR-0815, CUR-0820, CUR-0821 and CUR-0822 corresponds respectively to SEQ ID NOS:55,54,53,52,51 and the sample handled of 56-58.

Fig. 1 G is PCR in real time result's figure, shows to use compared with the control to use thiophosphate oligonucleotide that Lipofectamine 2000 introduces and handle the multiple variation+standard deviation in LCAT mRNA behind the Vero cell.PCR in real time result shows, 48 h after using at a processing in the oligomer of LCAT antisense Hs.668679 design, and the level of LCAT mRNA significantly increases in the Vero cell.The bar that is expressed as CUR-0819, CUR-0818, CUR-0817, CUR-0816, CUR-0815, CUR-0820, CUR-0821 and CUR-0822 corresponds respectively to SEQ ID NOS:55,54,53,52,51 and the sample handled of 56-58.

Fig. 1 H is PCR in real time result's figure, shows to use compared with the control to use thiophosphate oligonucleotide that Lipofectamine 2000 introduces and handle the multiple variation+standard deviation in LRP1 mRNA behind the HepG2 cell.PCR in real time result shows that at oligomer processing back 48 h that use at LRP1 antisense DC401271, the level of LRP1 mRNA significantly increases in the HepG2 cell.The bar that is expressed as CUR-0767 to CUR-0769, CUR-0771, CUR-0770, CUR-0775, CUR-0773, CUR-0772 and CUR-0774 corresponds respectively to SEQ ID NOS:59-61,63,62,67,65,64 and 66 samples of handling.

Fig. 1 I is PCR in real time result's figure, shows to use compared with the control to use thiophosphate oligonucleotide that Lipofectamine 2000 introduces and handle the multiple variation+standard deviation in LRP1 mRNA behind the Vero cell.PCR in real time result shows that at oligomer processing back 48 h that use at LRP1 antisense DC401271 and Hs.711951, the level of LRP1 mRNA significantly increases in the Vero cell.The bar that is expressed as CUR-0768, CUR-0767, CUR-0774 and CUR-0772 corresponds respectively to SEQ ID NOS:60,59,66 and 64 samples of handling.

Fig. 1 J is PCR in real time result's figure, shows to use compared with the control to use thiophosphate oligonucleotide that Lipofectamine 2000 introduces and handle the multiple variation+standard deviation in LRP1 mRNA behind the 3T3 cell.PCR in real time result shows that at oligomer processing back 48 h that use at LRP1 antisense DC401271 and AW544265, the level of LRP1 mRNA significantly increases in the 3T3 cell.The bar that is expressed as CUR-1017 to CUR-1022 corresponds respectively to the sample of handling with SEQ ID NOS:68-73.

Fig. 1 K is PCR in real time result's figure, shows to use compared with the control to use thiophosphate oligonucleotide that Lipofectamine 2000 introduces and handle the multiple variation+standard deviation in LDLR mRNA behind the HepG2 cell.PCR in real time result shows that at antisense scant polymer processing back 48 h that use at LDLR antisense sherflor.aApr07, the level of LDLR mRNA significantly increases in the HepG2 cell.The bar that is expressed as CUR-1054 to CUR-1059 corresponds respectively to the sample of handling with SEQ ID NOS:74-79.

Fig. 1 L is PCR in real time result's figure, shows to use compared with the control to use thiophosphate oligonucleotide that Lipofectamine 2000 introduces and handle the multiple variation+standard deviation in LDLR mRNA behind the HepG2 cell.PCR in real time result's demonstration, after the antisense scant polymer processing of using at LDLR antisense bloflor.aApr07, the level of LDLR mRNA in the HepG2 cell.The bar that is expressed as CUR-1059 to CUR-1063 corresponds respectively to the sample of handling with SEQ ID NOS:79-83.

Fig. 1 M is PCR in real time result's figure, shows to use compared with the control to use thiophosphate oligonucleotide that Lipofectamine 2000 introduces and handle the multiple variation+standard deviation in ApoE mRNA behind the HepG2 cell.PCR in real time result shows, 48 h after using at three processing in the antisense scant polymer of ApoE antisense Hs.626623 design, and the level of APOE mRNA significantly increases in the HepG2 cell.The bar that is expressed as CUR-0978, CUR-0980, CUR-0981, CUR-0979, CUR-0973, CUR-0975, CUR-0974, CUR-0977 and CUR-0976 corresponds respectively to SEQ ID NOS:89,91,92,90,84,86,85,88 and 87 samples of handling.

Fig. 1 N to Fig. 1 P represents PCR in real time result's figure, shows to use compared with the control to use thiophosphate oligonucleotide that Lipofectamine 2000 introduces and handle the multiple variation+standard deviation in ApoA1 mRNA behind the HepG2 cell.PCR in real time result shows, is using at some processing back 48 h in the antisense scant polymer of ApoA1 antisense DA327409ext, and the level of ApoA1 mRNA significantly increases in the HepG2 cell.Bar RH3-RH597 corresponds respectively to the sample of handling with SEQ ID NOS:171-248.

Fig. 1 Q is PCR in real time result's figure, show and compare, after handling the HepG2 cell during 7 days, at the last figure of ApoA1 mRNA(with naked LNA or thiophosphate oligonucleotide) and ApoA1 natural antisense DA327409ext RNA(figure below) in the multiple variation.The bar that is designated as #6LNA, #11LNA, #6PS and #11PS is represented SEQ ID NO:249-252 respectively.

Fig. 1 R is PCR in real time result's figure, be presented at handle the HepG2 cell with the LNA oligonucleotide after, orange of ApoA1 mRNA() and ApoA1 natural antisense DA327409ext RNA(blue bar) in the multiple variation.The bar that is designated as 6-11 is corresponding to SEQ ID NOS 249,257-260 and 250.

After Fig. 1 S is presented at and handles the HepG2 cell with oligonucleotide, at ApoA1 mRNA(figure below) and protein (last figure) in the dose dependent increase.The bar of indication CUR-4806 and CUR-4811 corresponds respectively to SEQ ID NOS 249 and 250.

Fig. 1 T is PCR in real time result's figure, be presented at use handle at the oligonucleotide of natural A poA1 antisense DA327409ext after, the rise of ApoA1 mRNA in former generation cercopithecus aethiops hepatocyte.The bar of indication CUR-4816 and CUR-4811 corresponds respectively to SEQ ID NOS:263 and 250.

Fig. 1 U shows as measuring by PCR in real time and ELISA respectively, compare with baseline values, with the oligonucleotide of CUR-962(at ApoA1 antisense DA327409ext design) handle after, ApoA1 mRNA that in the monkey liver biopsy, increases and the figure of protein level (right figure).After the identical time period, ApoA1 mRNA or protein level do not change (left figure) in being used in external demonstration the ApoA1 level is not had the matched group of oligonucleotide (CUR-963) administration of effect.The bar of indication CUR-962 and CUR-963 corresponds respectively to SEQ ID NOS:260 and 261.

Fig. 1 V shows the position of comparing side by side and being used for some oligonucleotide of these sequences of targeting of people and Rhesus Macacus ApoA1 natural antisense sequence.

Fig. 2 shows

SEQ ID NO:1: homo sapiens's ATP-binding cassette, subfamily A (ABC1), member 1 (ABCA1), mRNA (NCBI registration number: NM_005502)

SEQ ID NO:2: homo sapiens's Lecithin-cholesterol acyltransferase. (LCAT), mRNA (NCBI registration number: NM_000229.1)

SEQ ID NO:3: homo sapiens low density lipoprotein receptor relative protein 1 (LRP1), mRNA (NCBI registration number: NM_002332.2)

SEQ ID NO:4: house mouse LDH receptor related protein 1 (Lrp1), mRNA (NCBI registration number: NM_008512.2)

SEQ ID NO:5: homo sapiens low density lipoprotein receptor (LDLR), mRNA (NCBI registration number: NM_000527.3)

SEQ ID NO:6: homo sapiens's apo E (APOE), mRNA (NCBI registration number: NM_000041.2)

SEQ ID NO:7: homo sapiens's apolipoprotein A-1 (APOA1), mRNA (NCBI registration number: NM_000039).

Fig. 3 shows

SEQ ID NO:8: the natural ABCA1 antisense sequences of people (AK311445)

SEQ ID NO:9: mice natural A BCA1 antisense sequences (BF133827)

SEQ ID NO:10: the natural LCAT antisense sequences of people (Hs.668679)

SEQ ID NO:11: the natural LCAT antisense sequences of people (Hs.593769)

SEQ ID NO:12: the natural LCAT antisense sequences of people (Hs.387239)

SEQ ID NO:13: the natural LRP1 antisense sequences of people (Hs.711951)

SEQ ID NO:14: the natural LRP1 antisense sequences of people (DC401271)

SEQ ID NO:15: the natural LRP1 antisense sequences of people (BM933147)

SEQ ID NO:16: the natural LRP1 antisense sequences of mice (CK626173)

SEQ ID NO:17: the natural LRP1 antisense sequences of mice (AW544265)

SEQ ID NO:18: the natural ABCA1 antisense sequences of people (bloflor.aApr07)

SEQ ID NO:19: the natural ABCA1 antisense sequences of people (sherflor.aApr07)

SEQ ID NO:20: natural A POE antisense sequences (Hs.626623)

SEQ ID NO:21: natural A POE antisense sequences (Hs.714236)

SEQ ID NO:22: natural A POA1 antisense sequences (DA327409 of prolongation).

Fig. 4 shows ABCA1 antisense oligonucleotide SEQ ID NOs:23 to 40.' r ' indication RNA and * indication thiophosphate (phosphothioate) key.

Fig. 5 shows LCAT antisense oligonucleotide SEQ ID NOs:41 to 58.' r ' indication RNA and * indication phosphorothioate bond.

Fig. 6 shows LRP1 antisense oligonucleotide SEQ ID NOs:59 to 73.* indicate phosphorothioate bond.

Fig. 7 shows LDLR antisense oligonucleotide SEQ ID NOs:74 to 83.* indicate phosphorothioate bond.

Fig. 8 shows ApoE antisense oligonucleotide SEQ ID NOs:84 to 92.* indicate phosphorothioate bond.

Fig. 9 shows ApoA1 antisense oligonucleotide SEQ ID NOs:93 to 263.' r ' indication RNA and * indication phosphorothioate bond.

Figure 10 shows ABCA1 antisense oligonucleotide SEQ ID NOs:264 to 266.Antisense oligonucleotide SEQ ID NO:264 to 266 is respectively the reverse complemental body of antisense oligonucleotide SEQ ID NO:23 to 24.' r ' indicates RNA.

Figure 11 shows LCAT antisense oligonucleotide SEQ ID NOs:275 to 282.Antisense oligonucleotide SEQ ID NO:267 to 274 is respectively the reverse complemental body of antisense oligonucleotide SEQ ID NO:41 to 48.' r ' indicates RNA.

Figure 12 shows

SEQ ID NOs:275 to 277: correspond respectively to probe sequence, forward primer sequence and reverse primer sequence about custom design analysis to ApoA1 antisense DA327409ext,

SEQ ID NO:278: corresponding to CUR 962, * indication phosphorothioate bond and+indication LNA.

SEQ ID NO:279: corresponding to CUR 963, * indication phosphorothioate bond and+indication LNA.

Detailed Description Of The Invention

Several aspect of the present invention has hereinafter been described with regard to being used for illustrational example application.Be to be understood that numerous details, relation and the method set forth, so that overall understanding of the present invention to be provided.Yet the person of ordinary skill in the relevant will recognize easily that the present invention can need not one or more details or puts into practice with additive method.The present invention is not moved or event ordering limits, because some actions can take place simultaneously with different order and/or with other actions or incident.In addition, be not to need all illustrational actions or incident to realize according to method of the present invention.

All genes disclosed herein, gene title and gene outcome expection are corresponding to the homologue from any species, and compositions disclosed herein and method can be applicable to described any species.Therefore, this term includes but not limited to gene and the gene outcome from people and mice.Be to be understood that when disclosing from the gene of specific species or gene outcome, this disclosure expection only is exemplary, and is not interpreted as restriction, unless the context that it occurs therein spells out.Therefore, for example, for gene disclosed herein, this relates to mammalian nucleic acid and aminoacid sequence in some embodiments, expection comprises homology and/or orthologous gene and the gene outcome from other animals, includes but not limited to other mammals, Fish, amphibian, reptiles and birds.In preferred embodiments, gene or nucleotide sequence are the people.

Definition

Term used herein only is used to describe the purpose of specific embodiments, and does not expect restriction the present invention.As used herein, singulative " ", " a kind of " and " being somebody's turn to do " also expect and comprise plural form, unless context offers some clarification in addition.In addition, with regard to term " comprise ", " having ", " containing " or its variation be used for describing in detail and/or claim, this type of term expection is to be similar to the comprising property of mode that term " comprises ".

Term " about " or " approximately " mean as determining by those of ordinary skills, and in the acceptable error scope about occurrence, this will partly depend on this value and how measure or measure, i.e. the limitation of measuring system.For example, " pact " can mean according to the practice of this area 1 or surpass in 1 standard deviation.Alternately, " pact " can mean up to set-point 20%, preferably up to 10%, more preferably up to 5% and more preferably up to 1% scope.Alternately, with regard to biosystem or process, this term can mean in an order of magnitude of value, preferably in 5 times, and more preferably in 2 times especially.When occurrence is described, except as otherwise noted, otherwise should suppose that term " about " means in the acceptable error scope about occurrence in the application and claim.

As used herein, term " mRNA " means one or more mRNA transcripies of present known target gene, and any further transcript that can illustrate.

" antisense oligonucleotide " or " antisense compounds " means and another kind of RNA or DNA(target RNA, DNA) bonded RNA or dna molecular.For example, if it is the RNA oligonucleotide, it interacts by means of RNA-RNA and combines with another kind of RNA target so, and the activity of change target RNA (people such as Eguchi, (1991) Ann. Rev. Biochem. 60,631-652).Antisense oligonucleotide can raise or reduce the expression and/or the function of specific polynucleotide.This definition is intended to comprise from treatment, diagnosis or other viewpoints it seems useful any foreign rna or dna molecular.This quasi-molecule comprise antisense RNA for example or dna molecular, RNA interfering (RNAi), Microrna, bait RNA molecule, siRNA, enzymatic RNA, treatment editor RNA and agonist and antagonist RNA, antisense oligomeric compounds, antisense oligonucleotide, external guide sequence (EGS) oligonucleotide, alternative splicing thing, primer, probe and with other oligomeric compounds of target nucleic acid to small part hybridization.Like this, these chemical compounds can be introduced with the form of strand, two strands, part strand or cyclic oligomeric chemical compound.

In background of the present invention, term " oligonucleotide " refers to the oligomer or the polymer of ribonucleic acid (RNA) or DNA (deoxyribonucleic acid) (DNA) or its analogies.Term " oligonucleotide " also comprises linearity or cyclic oligomeric thing natural and/or modification monomer or bonding, comprises dezyribonucleoside, ribonucleotide, its displacement and α-anomer form, peptide nucleic acid(PNA) (PNA), lock nucleic acid (LNA), thiophosphate, methyl phosphonate etc.Oligonucleotide can combine with the target polynucleotide specificity by the regular pattern of monomer interphase interaction, for example Wo Sen-Ke Like (Watson-Crick) type base pairing, Ho gsteen or reverse Ho gsteen type base pairing etc.

Oligonucleotide can be " chimeric ", promptly is made up of zones of different.In background of the present invention, " chimeric " chemical compound is an oligonucleotide, and it comprises 2 or more a plurality of chemical regions, for example one or more DNA zone, one or more RNA zone, one or more PNA zone etc.Each chemical regions is made of at least one monomer unit, i.e. nucleotide under the situation of oligonucleotide chemical compound.These oligonucleotide generally comprise at least one zone that oligonucleotide is wherein modified, so that demonstrate one or more required character.The required character of oligonucleotide for example includes but not limited to that the resistance to nuclease degradation increases, cellular uptake increases and/or increases for the binding affinity of target nucleic acid.Therefore the zones of different of oligonucleotide can have heterogeneity.The mixed structure that chimeric oligonucleotide of the present invention can be used as two or more aforesaid oligonucleotide, modified oligonucleotide, oligonucleoside and/or oligonucleotide analogs forms.

Oligonucleotide can be by forming with the zone that " recorder (register) " connects, and promptly when monomer connects continuously, as in n DNA, or connects via sept.The sept expection constitutes the covalency " bridge " between the zone, and has the length that is no more than about 100 carbon atoms in the preferred case.Sept can carry different functionalities, for example has the plus or minus electric charge, carries specific nucleic acid in conjunction with character (intercalating agent, groove binder, toxin, fluorogen etc.), is lipophilic, induces specific secondary structure to contain the alanine peptide as what for example induce alpha-helix.

As used herein, " lipid transfer and metabolic gene " comprises all family members, mutant, allele, fragment, kind, coding and non-coding sequence, justice and antisense polynucleotides chain etc. arranged.

As used herein, term ATP-binding cassette 1, lipid transfer and metabolic gene, abc transport albumen 1, cholesterol flow out and regulate albumen (CERP), ABCA1, ABC-1, ABC1, CERP, FLJ14958, HDLDT1, the interchangeable in this application application of TGD.

As used herein, the interchangeable in this application application of term lecithin cholesterol acyltransferase, LCAT, phosphatidylcholine-sterol acyltransferase, phospholipid-cholesterol acyltransferase.

As used herein, term A2MR, α-2-macroglobulin receptor, APOER, apo E receptor, APR, CD91, FLJ16451, IGFBP3R, LRP, LRP-1, MGC88725, preceding low density lipoprotein receptor-associated protein 1, the interchangeable in this application application of TGFBR5.

As used herein, the interchangeable in this application application of term LDLR, FH, FHC, LDLCQ2, ldl receptor, low density lipoprotein receptor.

As used herein, the interchangeable in this application application of term AD2, Apo-E, ApoE, apo E, LDLCQ5, LPG, MGC1571.

As used herein, term ApoA1, Apo-A1, ApoA 1, the interchangeable in this application application of MGC117399.

As used herein, term " right ... as to have specific oligonucleotide " or " targeting ... oligonucleotide " oligonucleotide that refers to have following sequence: (i) can form stable compound, or (ii) can form stable duplex with the part of the mRNA transcript of target gene with the part of target gene.The stability of complex and duplex can be measured by Theoretical Calculation and/or external test.Stability exemplary that is used for measuring hybridization complex and duplex is determined at hereinafter, and embodiment describes.

As used herein, term " target nucleic acid " comprises DNA, the RNA(that transcribed by this class DNA comprises mRNA precursor and mRNA) and derived from cDNA, coding, the non-coding sequence of this type of RNA, justice or antisense polynucleotides are arranged.The normal function of the specific hybrid interfere RNA of oligomeric compounds and its target nucleic acid.By this target nucleic acid function adjusting of the chemical compound of specific hybrid with it, be commonly referred to as " antisense ".Treat that interferential DNA function comprises and for example duplicate and transcribe.Treat interferential RNA function comprise all great functions for example the RNA transposition to the protein translation site, by RNA translated protein, RNA montage catalytic activity to obtain one or more mRNA kinds and can be connected with RNA or promote by RNA.The interferential population effect of this type of target nucleic acid function is to regulate the expression of coded product or oligonucleotide.

RNA disturbs " RNAi " numerator mediated by double-stranded RNA (dsRNA), and itself and its " target " nucleotide sequence has the sequence-specific homology, and (Caplen, N. J. waits the people, (2001) Proc. Natl. Acad. Sci. USA 98:9742-9747).In particular of the present invention, instrumentality is 5-25 nucleotide " little interference " RNA duplex (siRNAs).(Bernstein, E. wait the people to siRNAs, (2001) derived from the dsRNA processing of the RNA enzyme by being called Dicer Nature409:363-366).SiRNA duplex product is recruited the inductive silencing complex of called after RISC(RNA) polyprotein matter siRNA complex in.Do not wish to be subjected to any concrete theory, the RISC target nucleic acid (suitably mRNA) that is considered to subsequently lead interacts in the sequence-specific mode at the siRNA of this place duplex, and (Bernstein, E. wait the people, (2001) with catalysis form mediation cutting Nature409:363-366; Boutla, A. waits the people, (2001) Curr. Biol. 11:1776-1780).Can operation well-known according to this area and that those of ordinary skill is familiar with synthetic and use can be according to the siRNA s of the present invention's use.The siRNA s that is used for using in the method for the invention suitably comprises about 50 nucleotide of about 1 – (nt).In the example of non-limiting embodiments, siRNAs can comprise about 5-Yue 40 nt, about 5-Yue 30 nt, about 10-Yue 30 nt, about 15-Yue 25 nt or about 20-25 nucleotide.

The selection of suitable oligonucleotide obtains promoting that described computer program is compared nucleotide sequence automatically and pointed out homogeneity or the homology zone by the program of using a computer.The nucleotide sequence that this class method is used for relatively obtaining is for example by search database GenBank or by order-checking PCR product for example.The nucleotide sequence that relatively allows to select to show the suitable homogeneity degree between the species from the nucleotide sequence of a series of species.Under the situation of the gene that does not check order, carry out southern blotting technique to allow to measure the homogeneity degree between gene in target species and other species.By under various strict degree, carrying out southern blotting technique, as known in the art, can obtain the approximate measure of homogeneity.Such oligonucleotide allow is selected in these operations, its demonstrate with experimenter to be controlled in the height of target nucleic acid sequence complementary and with other species in corresponding nucleic sequence than the low degree complementarity.The technical staff will recognize that there is sizable move place in the appropriate area at the gene of selecting to be used for using in the present invention.

" enzymatic RNA " means RNA molecule (Cech, (1988) with enzymatic activity J. American. Med. Assoc. 260,3030-3035).Enzymatic nucleic acid (ribozyme) works by at first combining with target RNA.This type of takes place in conjunction with the target bound fraction by enzymatic nucleic acid, and described enzymatic nucleic acid partly keeps closely approaching with the enzymatic of the molecule that acts on cutting target RNA.Therefore, enzymatic nucleic acid at first discern and subsequently by base pairing in conjunction with target RNA, and in case combine with correct site, with regard to enzymatic catalysis with cutting target RNA.

" bait RNA " means the RNA molecule of simulation about the natural binding structural domain of part.Therefore bait RNA competes the binding specificity part with the natural target that combines.For example, the overexpression that has shown HIV trans-activating response (TAR) RNA can serve as " bait " and effectively in conjunction with HIV tat protein, thus stop its combine with the TAR sequence of encoding among the HIV RNA (people such as Sullenger, (1990), Cell, 63,601-608).This is intended to is specific examples.The technical staff will recognize that this only is an example, and other embodiments can use the general known technology in this area easily to generate.

As used herein, the monomer that the general indication of term " monomer " connects by phosphodiester bond or its analog, individual to the oligonucleotide of hundreds of monomer unit approximately to form magnitude range from for example about 3-4 of several monomer unit.The analog of phosphodiester bond comprises: thiophosphate, phosphorodithioate, methyl phosphonate, seleno phosphate ester, phosphoramidate etc., and as hereinafter describing more comprehensively.

Term nucleotide " nucleotide " is contained the nucleotide of naturally occurring nucleotide and non-natural existence.For those skilled in the art should be clear and definite be before to be considered as " non-natural exists " various nucleotide to find at occurring in nature subsequently.Therefore, " nucleotide " not only comprises known purine-containing and pyrimidine heterocyclic molecule, also comprises its heterocyclic analogs and tautomer.The illustrative example of the nucleotide of other type is to comprise adenine, guanine, thymus pyrimidine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N ⁶-methyladenine, 7-denitrogenation xanthine, 7-deazaguanine, N ⁴, N ⁴-ethanol cytosine (ethanocytosin), N ⁶, N ⁶-ethanol-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)-people such as alkynyl cytosine, 5-fluorouracil, 5-bromouracil, false iso-cytosine, 2-hydroxy-5-methyl base-4-Triazolopyridine, iso-cytosine, isoguanine, inosine and Benner, U.S. Patent number 5, the molecule of " non-natural exists " nucleotide of describing in 432,272.Term " nucleotide " expection contain these examples with and analog and tautomer in each and all.Particularly advantageous nucleotide is to comprise those of adenine, guanine, thymus pyrimidine, cytosine and uracil, and this is regarded as the naturally occurring nucleotide relevant with diagnostic application with the treatment in the people.Nucleotide comprises natural 2'-deoxidation and 2'-hydroxyl sugar, for example as Kornberg and Baker, and DNA Replication, described in the second edition (Freeman, San Francisco, 1992), and their analog.

" analog " of mentioning nucleotide comprise the sugar moieties of base portion with modification and/or modification synthesizing ribonucleotide (referring to, for example by following general description: Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier and Altmann, (1997) Nucl. Acid. Res., 25(22), 4429-4443, Toulm é, J.J., (2001) NatureBiotechnology 19:17-18; Manoharan M., (1999) Biochemica et Biophysica Acta1489:117-139; Freier S. M., (1997) Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery ﹠amp; Development, 3:203-213, Herdewin P., (2000) Antisense ﹠amp; Nucleic Acid Drug Dev., 10:297-310); (referring to for example, N.K Christiensen. waits the people to [3.2.0] dicyclo arabinose nucleoside that 2'-O, 3`-C-connect, (1998) J. Am. Chem. Soc., 120:5458-5463; Prakash TP, Bhat B. (2007) Curr Top Med Chem.7 (7): 641-9; People such as Cho EJ. (2009) Annual Review of Analytical Chemistry, 2,241-264).This type of analog comprises the synthetic nucleosides that is designed to strengthen in conjunction with character, and is described in conjunction with character for example duplex or triplex stability, specificity etc.

As used herein, " hybridization " mean the pairing of the complementary strand basically of oligomeric compounds.A kind of mechanism of matching relates to the hydrogen bonding between the complementary nucleoside of oligomeric compounds chain or nucleotide base (nucleotide), and this can be Wo Sen-Ke Like, Ho gsteen or reverse Ho gsteen hydrogen bonding.For example, adenine and thymine is paired complementary nucleotide by forming hydrogen bond.Hybridization can take place in all cases.

The normal function that disturbs target nucleic acid when combining of chemical compound and target nucleic acid is when causing function and/or active the adjusting, and the complementarity that has enough degree, needing therein to avoid the non-specific binding of antisense compounds and non-target nucleic acid sequence under the bonded condition of specificity, promptly measure in vivo or the therapeutic treatment situation under under physiological condition, wherein carrying out under the condition of measuring under the external test situation, antisense compounds is " but specific hybrid ".

As used herein, phrase " stringent hybridization condition " or " stringent condition " refer to its down chemical compound of the present invention will be with its target sequence hybridization but with the condition of other sequence hybridizations of bottom line number.Stringent condition is a sequence dependent, and in different situations with will be different in background of the present invention, its down " stringent condition " of oligomeric compounds and target sequence hybridization by the character of oligomeric compounds with form and they treat that the mensuration of studying therein determines.Generally speaking, stringent hybridization condition comprises having for example Na of inorganic cation ⁺⁺Or K ⁺⁺The low concentration of (being low ionic strength) (＜0.15M) salt, be higher than 20 ℃-25 ℃ and be lower than oligomeric compounds: the temperature of the Tm of target sequence complex and denaturant be the existence of Methanamide, dimethyl formamide, dimethyl sulfoxide or detergent sodium lauryl sulphate (SDS) for example.For example, hybridization speed descends 1.1% for per 1% Methanamide.The example of high stringent hybridization condition is 60 ℃ of following 0.1X sodium chloride-sodium citrate buffers (SSC)/0.1%(w/v) SDS totally 30 minutes.

As used herein, " complementarity " refers to about accurate paired ability between 2 nucleotide on or two the oligomerization chains.For example, if the nuclear base on the ad-hoc location of antisense compounds can with the nuclear base hydrogen bonding on the ad-hoc location at target nucleic acid, described target nucleic acid is DNA, RNA or oligonucleotide molecules, and the position of hydrogen bonding is regarded as complimentary positions between oligonucleotide and target nucleic acid so.When every kind of enough purpose complimentary positions of molecule mesopodium when the nucleotide of hydrogen bonding occupies each other, oligomeric compounds and DNA, RNA or oligonucleotide molecules are complimentary to one another further.Therefore, " but specific hybrid " and " complementary " are accurate pairing or the complementary terms that is used to the enough degree on the nucleotide of enough numbers of pointing out, thereby make stable and specificity is combined between oligomeric compounds and the target nucleic acid and takes place.

But this area be to be understood that the sequence of oligomeric compounds need not with its target nucleic acid 100% complementation to be specific hybrid.In addition, oligonucleotide can be hybridized on one or more sections, thereby makes insertion or adjacent segments not relate to hybridisation events (for example, ring structure, mispairing or hairpin structure).Oligomeric compounds of the present invention comprise with target region in its target nucleic acid sequence of their targeting at least about 70% or at least about 75% or at least about 80% or at least about 85% or at least about 90% or at least about 95% or at least about 99% sequence complementarity.For example, wherein 18 in 20 of antisense compounds nucleotide are complementary and therefore with the antisense compounds of specific hybrid with target region, will represent 90% complementarity.In this example, all the other incomplementarity nucleotide can cluster or are interrupted by complementary nucleotide, and with each other or complementary nucleotide to need not be adjacency.Like this, having 4(four) antisense compounds of 18 nucleotide of length of individual incomplementarity nucleotide will have 77.8% overall complementary with target nucleic acid, and therefore will fall within the scope of the present invention, described incomplementarity nucleotide side is 2 zones complementary fully with target nucleic acid.Use blast program known in the art (base local comparison research tool) and PowerBLAST program (people such as Altschul, (1990) J. Mol. Biol., 215,403-410; Zhang and Madden, (1997) Genome Res., 7,649-656), can more solito measure the complementary percentage ratio in antisense compounds and target nucleic acid zone.Percent homology, sequence homogeneity or complementarity can be by for example Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) use default setting to measure, the algorithm of described Gap program use Smith and Waterman ( Adv. Appl. Math., (1981) 2,482-489).

As used herein, term " pyrolysis chain temperature (Tm) " refers in the temperature that limits under ionic strength, pH and the nucleic acid concentration, hybridizes with target sequence in poised state with 50% oligonucleotide of target complement sequence down at it.Usually, stringent condition will be for short oligonucleotide (for example, 50 nucleotide of 10 –), and wherein salinity is to be at least about those of 30 ℃ pH 7.0-8.3 times at least about 0.01-1.0 M Na ion concentration (or other salt) and temperature.Stringent condition can also with add destabilizing agent for example Methanamide reach.

As used herein, " adjusting " mean the increase (stimulation) in the gene expression or reduce (inhibition).

When using in the background at polynucleotide sequence, term " variant " can comprise the polynucleotide sequence relevant with wild type gene.This definition for example can also comprise " allele ", " montage ", " species " or " polymorphism " variant.Splice variant can have remarkable homogeneity with reference molecule, but because the alternative splicing of exon in the mRNA course of processing, generally will have the polynucleotide of more or less number.Corresponding polypeptide can have not existing of other functional domain or domain.The species variant is to another different polynucleotide sequence from species.What have special effectiveness in the present invention is the variant of wild type gene product.Variant can result from least one sudden change in the nucleotide sequence, and may cause changing mRNAs or its structure or function and may change or immovable polypeptide.Any given natural or recombination can have none, one or more allelic form.The routine sudden change that causes variant changes natural disappearance, interpolation or the displacement that generally belongs to nucleotide.These type changes separately can be separately or are combined in other one or many takes place in the given sequence.

Resulting polypeptide generally will have relative to each other significant aminoacid homogeneity.The polymorphism variant is the variation in the polynucleotide sequence of specific gene between the individuality of given species.The polymorphism variant can also comprise " single nucleotide polymorphism " (SNPs) or wherein polynucleotide sequence by the single base mutation of a sequence change.The existence of SNPs can be indicated the special group that for example has tendency for morbid state, i.e. susceptibility and resistance are relatively.

The polynucleotide of deriving comprise the nucleic acid of implementing chemical modification, for example replace hydrogen by alkyl, aryl or amino group.Derivant for example derivatized oligonucleotide can comprise the part that non-natural exists, for example bonding between sugar moieties of Gai Bianing or sugar.Exemplary in these is thiophosphate and other sulfur-bearing kinds known in the art.Derivative nucleic acids can also comprise labelling, comprises ribonucleotide, enzyme, fluorescent agent, chemiluminescence agent, developer, substrate, cofactor, inhibitor, magnetic-particle etc.

" derive " polypeptide or peptide is for example to handle modify the sort of by glycosylation, Pegylation, phosphorylation, sulphation, reduction/alkylation, acidylate, chemical coupling or weak formalin.Derivant can also directly or indirectly be modified, and to comprise detectable label, includes but not limited to radiosiotope, fluorescence and enzyme labelling.

As used herein, term " animal " or " patient " are intended to comprise for example people, sheep, elk, deer, mule deer, ermine, mammal, monkey, horse, cattle, pig, goat, dog, cat, rat, mice, birds, chicken, reptiles, Fish, insecticide and arachnidea.

" mammal " contains general homoiothermic animal (for example people and performing animal) under medical treatment and nursing.Example comprises cat, dog, horse, cattle and people, and people only.

" treatment " or " processing " contains the treatment of morbid state in the mammal, and comprises: (a) morbid state in the prevention mammal takes place, and particularly has at that time when this type of mammal has the morbid state tendency but is not diagnosed as yet; (b) suppress morbid state, for example stop its development; And/or (c) alleviate morbid state, for example impel morbid state to disappear up to reaching required end of the final point.Treatment also comprises the improving of disease symptoms (for example ease the pain or uncomfortable), and wherein this type of improvement can directly influence disease (for example, cause, infection, expression etc.) or can be not like this.

As used herein, " cancer " refers to include but not limited to all types of cancers or tumor or the malignant tumor found in the mammal: leukemia, lymphoma, melanoma, cancer and sarcoma.Cancer itself shows as " tumor " or comprises the tissue of pernicious cancerous cell.The example of tumor comprises cancer and sarcoma, such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchiogenic cancer, renal cell carcinoma, hepatoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, nephroblastoma, cervical cancer, tumor of testis, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neurocytoma and retinoblastoma.Can include but not limited to for example Hokdkin disease by other cancer according to combination treatment disclosed by the invention, non_hodgkin lymphoma, multiple myeloma, neurocytoma, breast carcinoma, ovarian cancer, pulmonary carcinoma, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinaemia, small cell lung cancer, primary brain tumors, gastric cancer, colon cancer, pernicious pancreas insulinoma (insulanoma), carcinoid malignant, bladder cancer, preceding dermatosis cancerates, carcinoma of testis, lymphoma, thyroid carcinoma, neurocytoma, the esophageal carcinoma, genitourinary cancer, pernicious hypercalcemia, cervical cancer, carcinoma of endometrium, adrenocortical carcinoma and cancer of pancreas.

" sacred disease or disease " refers to any disease or the disease of nervous system and/or visual system." sacred disease or disease " comprises disease or the disease that involves central nervous system's (brain, brain stem and cerebellum), peripheral nervous system (comprising cranial nerve) and autonomic nervous system (its part is arranged in maincenter and peripheral nervous system).The example of nervous disorders includes but not limited to headache, numb and stupor, dementia, epilepsy, sleep disorder, wound, infection, tumor, neural ophthalmology, the dyskinesia, demyelination, spinal cord disease and peripheral nerve, muscle and neuromuscular junction disease.Addiction and mental illness include but not limited to bipolar affective disorder and schizophrenia, and are also included within the definition of nervous disorders.Following is the several nervous disorders that can use according to the compositions and methods of the invention treatment, symptom, sign and syndromic enumerating: acquired epileptic aphasia, acute disseminated encephalomyelitis, adrenoleukodystrophy, age-related macular degeneration, corpus callosum hypoplasia, agnosia, aicardi's syndrome, Alexander disease, alper's disease, alternating hemiplegia, vascular dementia, amyotrophic lateral sclerosis, anencephaly, Angelman syndrome, angiomatosis, anoxia, aphasia, apraxia, arachnoid cyst, arachnoiditis, the Anronl-Chiari deformity, arteriovenous malformotion, the Asperger syndrome, ataxia telangiectasia, the many moving obstacles of attention deficit companion, autism, dysautonomia, backache, batten's disease, behcets disease, Bell's palsy, optimum essential blepharospasm, optimum focal, amyotrophy, benign intracranial hypertension, binswanger, blepharospasm, Bloch Sulzberger syndrome, brachia plexus injury, brain abscess, brain injury, cerebroma (comprising glioblastoma multiforme), tumor of spinal cord, Brown-Se﹠1﹠quard syndrome, canavan's disease, carpal tunnel syndrome, causalgia, the central pain syndrome, central pontine myelinolysis, the head disease, cerebral aneurysm, cerebral arteriosclerosis, brain atrophy, cerebral gigantism, cerebral palsy, charcot-Marie-Tooth disease, the neuropathy of chemotherapy-induced and neuropathic pain, chiari's deformity, chorea, chronic inflammatory demyelinating polyneuropathy, chronic pain, chronic local pain syndrome, CLS, stupor comprises the persistence persistent vegetative state, facial diplegia,congenital, the cortex substrate is degenerated, cranial arteritis, craniosynostosis, creutzfeldt-Jacob disease, cumulative bad wound obstacle, Cush, acidophilia's cytomegalovirus infection, cytomegalovirus infection, dancing eye-dancing foot syndrome, red fertile two Cotards, the gloomy disease in road, the De Mosaier syndrome, dejerine-Klumpke paralysis, dull-witted, dermatomyositis, diabetic neuropathy, Schilder, dysautonomia, dysgraphia, dyslexia, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis, brain is outstanding, encephalotrigeminal angiomatosis, epilepsy, erb's palsy, essential tremor, Fabry, fahr's syndrome, faint, familial spastic paraplegia, FC, Fisher syndrome, friedreich's ataxia, frontotemporal dementia and other " tau pathological changes ", familial splenic anaemia, Gerstmann syndrome, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, guillain-Barre syndrome, the myelopathy that HTLV-1-is relevant, hallervorden-Spatz disease, head injury, headache, hemifacial spasm, hereditary spastic paraplegia, heredopathia atactica polyneuritiformis, herpes auris, herpes zoster, Pingshan Mountain syndrome, dementia that HIV is relevant and neuropathy (being also referred to as the neurological performance of AIDS), holoprosencephaly, Huntington Chorea and other polyglutamic amide repeat sick, hydranencephaly, hydrocephalus, hypercortisolism, anoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, baby's phytanic acid storage disease, baby Lei Fusumu disease, infantile spasm, inflammatory myositis, entocranial cyst, intracranial hypertension, the Joubert syndrome, the Keams-Sayre syndrome, the sick dancing eyes syndrome of Kennedy, cervical vertebrae synostosis, krabbe's disease, Ku-Wei disease, Kuru disease, myoclonic epilepsy, lambert-Eatom myasthenic syndrome, the Landau-Kleffner syndrome, side marrow (Wahlen Burger) syndrome, learning disability, Leigh disease, the Lennox-Gustaut syndrome, lesch-Nyhan syndrome, leukodystrophy, dementia with Lewy body, agyria, locked in syndrome, Lu Gelei creutzfeldt jakob disease (that is, motor neuron or amyotrophic lateral sclerosis), the lumbar intervertebral disc disease, Lyme disease-neurological sequela, Ma-Yue disease, macrencephaly (macrencephaly), macrencephaly (megalencephaly), Mai-Luo two Cotards, Meniere syndrome, meningitis, Menkes disease, metachromatic leukodystrophy, microcephaly's deformity, migraine, Miller Fisher syndrome, transient apoplexy, mitochondrial myopathy, mobius syndrome, single limb amyotrophy, motor neuron, abnormal vascular net at the bottom of the brain, mucopolysaccharidosis, multiple sclerotic dementia, multifocal motor neuropathy, multiple sclerosis and other demyelination diseases, multiple system atrophy companion postural hypotension, the p duchenne muscular dystrophy, myasthenia gravis, the demyelination Schilder, myoclonic encephalopathy of infant, myoclonus, myopathy, congenital myotonia, narcolepsy, neurofibromatosis, neuroleptic malignant syndrome, the neurological performance of AIDS, the neurological sequela of lupus, neuromyotonia, NCL, the neuron disease of dividing a word with a hyphen at the end of a line, NP, the O'Sullivan-McLeod syndrome, occipital neuralgia, occult spinal dysraphism sequence, the former syndrome in land for growing field crops, olivopontocerebellar atrophy, opsoclonus, optic neuritis, orthostatic hypotension, overuse syndrome, paraesthesia, neurodegenerative disease or disease (parkinson disease, the Heng Yandun disease, Alzheimer, amyotrophic lateral sclerosis (ALS), dull-witted, multiple sclerosis and other diseases and the disease relevant) with nerve cell death, eulenburg disease, tumor is outer sick, the paroxysmal outbreak, parry-Romberg syndrome, pelizaeus-Merzbacher disease, periodic paralysis, peripheral neuropathy, pain neuropathy and neuropathic pain, the persistence persistent vegetative state, pervasive developmental disorders, the light sneezing reflex, phytanic acid storage disease, pager's disease, pinched nerve, pituitary tumor, polymyositis, the hole brain, poliomyelitis later stage syndrome, postherpetic neuralgia, postinfectious encephalomyelitis, postural hypotension, PW, primary lateral sclerosis, prion disease, parry-Romberg syndrome, progressive multifocal leukoencephalopathy, progressive sclerosing poliodystrophy, benumb on the carrying out property nuclear, pseudotumor cerebri, Ramsay-HH (I type and II type), the Rasmussen encephalitis, reflex sympathetic dystrophy syndrome, refsum, the repeatable motion disease, the repeatability stress damage, restless legs syndrome, the retrovirus retrovirus myelopathy of being correlated with, Rett syndrome, Reye syndrome, chorea, sandhoff's disease, schilder's disease, Schizencephaly, septum pellucidum-hypoplasia of optic nerve, SBS, herpes zoster, orthostatic hypotension syndrome, sjogren syndrome, sleep apnea, sotos' syndrome, spasticity, spina bifida, spinal cord injury, tumor of spinal cord, Duchenne-Arandisease, the stiff man syndrome, apoplexy, sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, sydenham chorea, faint, syringomyelia, tardive dyskinesia, Tay Sachs disease, temporal arteritis, tethered cord syndrome, thomsen's disease, thoracic outlet syndrome, trigeminal neuralgia, Todd's paralysis, tourette's syndrome, transient ischemic attack, propagated spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremble, trigeminal neuralgia, the spastic paraparesis in the torrid zone, tuberous sclerosis, vascular dementia (multi-infarct dementia), vasculitis comprises temporal arteritis, hippel-Lindau disease, dorsolateral medullary syndrome, familial spinal muscular atrophy, west's syndrome, ACTS, williams syndrome, Wei Ersenshi disease and zellweger's syndrome.

" inflammation " refers to the systemic inflammatorome situation and with the migration of mononuclear cell, leukocyte and/or neutrophil cell with attract the situation of local correlation.The example of inflammation includes but not limited to result from following inflammation: causal organism (comprising gram positive bacteria, gram negative bacteria, virus, fungus and parasite such as protozoacide and anthelmintic) infects, transplant rejection (comprises the repulsion of solid organ such as kidney, liver, the heart, lung or cornea, and the bone marrow graft repulsion, comprise graft versus host disease (GVHD)) or local chronic or acute autoimmune or allergy.Autoimmune disease comprises: acute glomerulonephritis, rheumatoid or reactive arthritis, chronic glomerulonephritis, inflammatory bowel such as Crohn disease, ulcerative colitis and necrotizing enterocolitis, granulocyte input related syndromes, inflammatory dermatosis such as contact dermatitis, atopic dermatitis, psoriasis, systemic lupus erythematosus (sle) (SLE), autoimmune thyroiditis, multiple sclerosis, with the diabetes of some form, or wherein the attack that causes of experimenter's autoimmune system causes any other autoimmune state of pathologic disorganization.Allergy comprises allergic asthma, chronic bronchitis, acute and tardy hypersensitivity.Whole body inflammatory diseases state comprises and following relevant inflammation: (for example pour into behind wound, burn, the ischemic event again, thrombosis incident in the heart, brain, intestinal or the peripheral blood vessel comprises myocardial infarction and apoplexy), sepsis, ARDS or multiple organ dysfunction syndrome.Inflammatory cell is raised and is also occurred in the atheromatous plaque.Inflammation includes but not limited to non_hodgkin lymphoma, wegener granulomatosis, struma lymphomatosa, hepatocarcinoma, atrophy of thymus gland, chronic pancreatitis, rheumatoid arthritis, reactive lymphoid hyperplasia, osteoarthritis, ulcerative colitis, papillary carcinoma, Crohn disease, ulcerative colitis, acute cholecystitis, chronic cholecystitis, sclerosis (cirrhosis), chronic sialadenitis, peritonitis, acute pancreatitis, chronic pancreatitis, chronic gastritis, endometriosis, endometriosis, acute cervicitis, chronic cervicitis, lymphoid hyperplasia, multiple sclerosis, be secondary to the hypertrophy of idiopathic thrombocytopenic purpura, Primary IgA nephropathy, systemic lupus erythematosus (sle), psoriasis, emphysema, chronic pyelonephritis and chronic cystitis.

Cardiovascular disease or disease comprise and can cause ischemia or be poured into those diseases that cause by heart again.The cardiovascular tissue damage that example includes but not limited to atherosclerosis, coronary artery disease, granuloma myocarditis, chronic myocarditis (non-granuloma), primary hypertrophic cardiomyopathy, peripheral arterial disease (PAD), apoplexy, angina pectoris, myocardial infarction, caused by asystole, the cardiovascular tissue damage, the cardiogenic shock that cause by heart bypass and one skilled in the art will recognize that or involve the conditions associated of the dysfunction of heart or vascular system or tissue injury, especially but be not limited to the tissue injury relevant with the metabolic gene activation with lipid transfer.The CVS disease includes but not limited to atherosclerosis, granuloma myocarditis, myocardial infarction, is secondary to the myocardial fibrosis of valvular heart disease, does not have myocardial fibrosis, primary hypertrophic cardiomyopathy and the chronic myocarditis (non-granuloma) of blocking.

The endocrine system disease and the disease of " metabolic disease or disease " wide region comprise for example insulin resistant, diabetes, obesity, impaired glucose tolerance, high blood cholesterol, hyperglycemia, hyperinsulinemia, dyslipidemia and hyperlipemia.

Polynucleotide and oligonucleotide composition and molecule

Target

In one embodiment, target comprises the nucleotide sequence of lipid transfer and metabolic gene, and it unrestrictedly comprises related with lipid transfer and metabolic gene have justice and/or non-coding of antisense and/or coded sequence.

In one embodiment, target comprises the nucleotide sequence of ABCA1, and it unrestrictedly comprises related with the ABCA1 gene have justice and/or non-coding of antisense and/or coded sequence.

In one embodiment, target comprises the nucleotide sequence of LCAT, and it unrestrictedly comprises related with the LCAT gene have justice and/or non-coding of antisense and/or coded sequence.

In one embodiment, target comprises the nucleotide sequence of LRP1, and it unrestrictedly comprises related with the LRP1 gene have justice and/or non-coding of antisense and/or coded sequence.

In one embodiment, target comprises the nucleotide sequence of low density lipoprotein receptor (LDLR), and it unrestrictedly comprises related with LDLR have justice and/or non-coding of antisense and/or coded sequence.

In one embodiment, target comprises the nucleotide sequence of apolipoprotein (ApoA1), and it unrestrictedly comprises related with ApoA have justice and/or non-coding of antisense and/or coded sequence.Human apolipoprotein A-I (ApoA-I) is high density lipoprotein (the main protein component of HDL and lymph Chylomicron.In human plasma, 4 kinds of main circulation lipoproteins have been named: Chylomicron (CM), very low density lipoprotein (VLDL) (VLDL), low density lipoprotein, LDL (LDL) and high density lipoprotein (HDL).HDL involves the removal of cholesterol from surrounding tissue, and this is by transporting it into liver or other lipoproteins carry out.

ATP-binding cassette subfamily-A (ABCAI) member I ABCAI plays the cholesterol efflux pump in cytolipin removal approach.

ATP-binding cassette transport protein (ABC-transport protein) is the member of following protein superfamily, and it is one of maximum representative in the existing door of from the prokaryote to people all and the most ancient family.Abc transport albumen is such transmembrane protein, and its energy that utilizes adenosine triphosphate (ATP) hydrolysis to be to carry out some bioprocess, comprises that various substrates stride the film dystopy, and non-transhipment correlated process, as RNA translation and DNA reparation.They are transported various substrates and stride across epicyte and cell inner membrance, comprise metabolite, lipid and sterin and medicine.Protein is abc transport albumen according to the tissue typing of sequence and their one or more ATP-binding cassettes (ABC) domain.Abc transport albumen involves tumor opposing, cystic fibrosis, antibacterial multiple medicines patience and a series of other heritability human disease.

The embrane-associated protein of ABC1 gene code is the member of ATP-binding cassette (ABC) transport protein superfamily.The various molecules of ABC protein transport stride across epicyte and cell inner membrance.The ABC gene is divided into 7 kinds of different subfamilies (ABCA, MDRITAP, MRP, ALD, OABP, GCN20, White).This protein is the member of ABCA subfamily.The member of ABCA subfamily comprises the unique main ABC subfamily that is found in uniquely in the many cells eukaryotic cell.With cholesterol is its substrate, and this protein plays a part to play the cholesterol efflux pump in cytolipin removal approach.

In preferred embodiments, antisense oligonucleotide is used for prevention or treatment and ATP-binding cassette molecule diseases associated or disease.ATP-binding cassette transport protein ABCl (member 1 of human transport protein subfamily ABCA) is also referred to as cholesterol and flow out regulates albumen (CERP), and it is by the protein of ABCl gene code in the people.This transport protein is the main instrumentality of cellular cholesterol and phospholipid stable state.

ABCl is present in the high density lipoprotein (HDL), and its permission is removed excessive cholesterol and phospholipid from people's cell membrane.Because this protein needs in whole machine body, it synthesizes as 220-kDa protein omnipresence ground.It is present in higher amount and shuttles back and forth or involve in the tissue such as liver, small intestinal and fatty tissue in lipid turnover.

Act on that the ABCl transport protein is expressed or the factor of its post translational modification also is to involve its molecule of function subsequently, as fatty acid, cholesterol, also be cytokine and 3'5'-AMP.

Low density lipoprotein receptor-associated protein 1 (LPR1) is about 4544 aminoacid; 504575 Da.It is that the 85-kDa film is in conjunction with the heterodimer of carboxyl subunit with the non-covalent 515-kDa amino terminal subunit that is connected.Cell intracellular domain and MAFB interact.LPR1 sees in the complex with PID1IPCLI1, LRP1 and CUBNI.Interact with SNX17, PID1IPCLI1, PDGF, LRPAP1 and CUBN.Cell intracellular domain and SHC1, GULP1 and DABl interact.

LRP1 is the endocytosis receptor, and it involves endocytosis and involves the local metabolism of the complex between plasma clearance, activator of plasminogen and the endogenous inhibitor thereof of the remaining and activatory LRPAP1 (alpha2-macroglobulin) of the phagocytosis of apoptotic cell, early embryonic development, cytolipin stable state, Chylomicron.Be not wishing to be bound by theory, its scalable cell incident is as signal conduction in APP metabolism, the kinases dependent cell, the conduction of neuron calcium ion signal and neurotransmission.

High density lipoprotein (HDL) is picked out the extra cholesterol in the blood and is made it turn back to liver.Low density lipoprotein, LDL (or LDL) is the main transport protein of body inner cholesterol.But can cause atherosclerosis (tremulous pulse narrows down and hardens) through too many for many years LDL, and cause heart disease or heart attack.Ratio removes the decision of HDL cholesterol by the LDL cholesterol.For example, if the individual has the HDL cholesterol of 50 mg/dL and the LDL cholesterol of 150 mg/dL, HDL/LDL ratio will be 0.33 so.Purpose is HDL/LDL ratio to be kept surpass 0.3, and wherein desirable HDL/LDL ratio is to surpass 0.4.

HDL in liver and small intestinal as the dish type granule de novo synthesis of rich in proteins.The main apoprotein of HDL is apoA-I, apoA-II, apoC-I, apoC-II and apoE.Recently the HDL of Xing Chenging comprises few cholesterol and cholesteryl ester.In the accumulation in the heart of hdl particle neutral core, HDL is transformed into spherical hdl particle by its initial disc shaped by cholesteryl ester.Cholesterol by HDL by chyle particle residue part of V LDL residual fraction (being also referred to as intermediated-density lipoprotein or IDL) with directly accumulate by cell surface membrane.Cholesterol is by HDL relevant enzyme lecithin: the effect of cholesterol acyltransferase (" LCAT ") obtains esterification.For fatty acid is transferred to the LCAT of the C-3-OH group of cholesterol from lecithin (phosphatidylcholine), need with the interaction of the ApoA-I that on the HDL surface, finds.The accumulation of core cholesteryl ester changes newborn HDL into HDL2 and HDL3.Referring to people such as R. I. Levy, " The structure, function and metabolism of high-density lipoproteins:A status report, " Circulation, the 62nd volume, IV4-8 page or leaf (1980); With people such as D. I. Silverman, " High-density lipoprotein subfractions, " Am. J. Med., the 94th volume, 636-45 page or leaf (1993).

HDL separates from blood plasma by ultracentrifugation usually.Normal HDL density range is 1.063 g/mL-1.21 g/mL, and this roughly is divided into 2 scope HDL2(1.063 g/mL-1.125 g/mL) and HDL3(1.125 g/mL-1.21 g/mL).Recently, be immunoblotting and enzyme connection difference antibody mediated immunity determining adsorption subsequently by two-dimensional electrophoresis, identified the main granule of among the HDL 2 colony.One of these colonies comprise the granule with independent apoA-I, and the another kind of granule with apoA-I and apoA-II that comprises.The particulate relative scale of apoA-I is the highest in HDL2 part, and HDL3 mostly are combinations of apoA-I and apoA-II.Referring to people such as J. C. Fruchart, " Apolipoprotein A-containing lipoprotein particles:physiological role, quantification, and clinical significance, " Clin. Chem., the 38th volume, 793-7 page or leaf (1992); And B.F. people such as Asztalos, " Normolipidemic subjects with low HDL cholesterol levels have altered HDL subpopulations, " Arterioscler. Thromb. Vasc. Biol., the 17th volume, 1885-1893 page or leaf (1997).

Human apolipoprotein A-I(ApoA-I) is the main protein constituent of HDL and lymph Chylomicron.ApoA-I is mainly synthetic as precursor protein (preceding former apo A-I) in liver and small intestinal.Preceding former apo A-I cuts in cell, with apo A-I before forming, is secreted into the form in blood plasma and the lymph.In blood plasma, 6 aminoacid cut among the apo A-I in the past, to form ripe ApoA-I.

The single non-glycosylated polypeptide that ripe ApoA-I is made up of 243 aminoacid of known array.ApoA-I serves as the cofactor of blood plasma enzyme (lecithin cholesterol acyltransferase (LCAT)), is responsible for forming the most of cholesteryl esters in the blood plasma.The ApoA-I of minimizing level can cause the development of the disease and the coronary heart disease of blood plasma lipide movement system.Low-level ApoA-I and HDL have been shown as the strong risk factor of heart attack and other atherosclerotic blood vessel diseases.Referring to U.S. Patent number 5,059,528 and 6,258,596.

Apo E (ApoE) be see Chylomicron and with hepatocyte and peripheral cell on the apoprotein of the bonded intermediated-density lipoprotein of specific receptor (IDLs).Intermediated-density lipoprotein belongs to hdl particle family and is formed by the degraded of very low density lipoprotein (VLDL).IDL is one of 5 kinds of main lipoprotein set (Chylomicron, VLDL, IDL, LDL, HDL), and it can make fat and cholesterol move in the group water solution of blood flow.Apo E (ApoE) is important for the normal catabolism of the lipoprotein component that is rich in triglyceride.

APOE gene A poE mapping is in the chromosome 19 of gathering together with apoC l and apoC 2.ApoE is made of 4 exons and 3 introns, totally 3597 base pairs.In melanocyte, APOE gene expression can be passed through ommatidium-dependency transcription factor (MITF) and regulate.This gene is polymorphic, has 3 main allele ApoE2, ApoE3, ApoE4, and it translates into proteinic 3 isotypes: normal-ApoE-E3; Dysfunction-ApoE-E2 and ApoE-E4.These isotypes only differ from one another by an aminoacid replacement in post-11.2 and 158.

Lecithin cholesterol acyltransferase (LCAT) is hepatogenous blood plasma enzyme, and cholesterol is to the conversion of cholesteryl ester on the catalysis lipoprotein, and it is by the fatty acid transacylateization of 3-hydroxyl on from phosphatidylcholine sn-2 position to cholesterol A-ring.Most of LCAT activity sees on the high density lipoprotein (HDL), but about 30% also on the lipoprotein that contains apolipoprotein (Apo) B.

Apolipoprotein E gene is polymorphic, has 3 main allele ApoE2, ApoE3, ApoE4.E2 and hereditary disease III type hyperlipoproteinemia and relevant with the risk of arteriosclerotic increase and reduction.E3 sees in about 64% the colony.Consider " neutrality " Apo E genotype.E4 helps also that arteriosclerosis and Alzheimer are sick now, cognitive function is impaired and axon growth weakens.

LCAT promotes the reverse cholesterol transport approach, and by this approach, excessive cellular cholesterol returns liver and drains.Be not wishing to be bound by theory, mechanism for example comprises: LCAT increases the HDL level, and HDL itself can increase cholesterol flowing from cell by the amount that increases cholesterol extracellular receptor.Simultaneously, but cholesterol is gone up LCAT by HDL esterification restricted cholesterol from the cytotropic spontaneous exchange backward of HDL and promote HDL to go up the clean conveying of cholesterol to liver.

In preferred embodiments, disease or the disease that antisense oligonucleotide is used to prevent or treatment is relevant with the metabolic gene family member with lipid transfer.Available from the cell/tissue treatment that utilizes the stem cell regenerating that antisense compounds obtains exemplary lipid transfer and metabolic gene is disease mediated and disease comprises: cardiovascular disease or disease, metabolic disease or disease (diabetes for example, fat, dyslipidemia, hyperlipidemia, hyperinsulinemia, hypercholesterolemia etc.), with impaired diseases associated of lipid metabolism or disease, coronary artery disease, arteriosclerosis, HDL metabolic disease or disease are (for example, Familial HDL deficiency (FHD), sea blue histiocyte syndrome, Tangier, fish eye disease, LCAT lacks, low HDL hypercholesterolemia etc.), with cellular cholesterol and/or phospholipid stable state diseases associated or disease, familial amyloid sample nephropathy, with impaired diseases associated of cholesterol regulation or disease, lack diseases associated or disease with lipid transfer and metabolic gene transport protein, apolipoprotein A-1 lacks, flow out diseases associated or disease with cholesterol in unusual fast or unusual at a slow speed the cell, with pancreatic beta cell function diseases associated or disease, diabetes, metabolic disease or disease, arthritis, inflammation, autoimmune disease or disease, acquired immune deficiency syndrome (AIDS) (AIDS), inflammation, sacred disease or disease, neurodegenerative disease or disease, cancer, dyslipidemia, metabolism syndrome, senile plaque, cerebral amyloid angiopathy, amyloidosis, glioblastoma, with amyloid deposition diseases associated or disease, neurofibrillary tangles, choriocarcinoma, astrocytoma, amyloidosis, hyperlipemia, tumor transforms, atheromatous plaque, block, shift, pulmonary fibrosis, necrosis, shock, melanoma, genetic predisposition, psoriasis, glioma, europathology, angiopathy, cell injury, nonsmall-cell lung cancer (NSCLCs), liposarcoma, immune deficiency disorder or disease, the organ-graft refection, allergy, glomerulonephritis, phlebothrombosis, pathological process or leukemia, skeletal diseases or disease, muscle disease or disease, with infection biological diseases associated or disease, immune correlated disease or disease, nervous lesion and paralysis, NE differentiation, the non-neuropathy amyloidosis of systematicness, amyloid disease depends on the tumor growth that blood vessel takes place, and non-Cancerous disease companion comprises that the symptom that increases takes place blood vessel, psoriasis for example, retinopathy of prematurity, choroidopathy, neovascular glaucoma, diabetic retinopathy, substance abuse, the impaired and axon growth minimizing of cognitive function, compare the ApoE unconventionality expression with normal control, function, active, psoriasis, the biological as virus of external source, antibacterial, parasite, fungus-caused disease or disease, etc.

In a preferred embodiment, lipid transfer and metabolic gene antisense oligonucleotide are used for organ transplantation (for example, liver transplantation, renal transplantation, bone marrow transplantation, heart transplantation etc.) in treatment.

In a preferred embodiment, oligonucleotide is special for the polynucleotide of lipid transfer and metabolic gene, and it includes but not limited to noncoding region.Lipid transfer and metabolic gene target comprise the variant of lipid transfer and metabolic gene; The mutant of lipid transfer and metabolic gene comprises SNPs; The non-coding sequence of lipid transfer and metabolic gene; Allele, fragment etc.Preferably, oligonucleotide is an antisense rna molecule.

According to embodiment of the present invention, target nucleic acid molecule is not limited to independent lipid transfer and metabolic gene polynucleotide, also extends to any in the isotype, receptor, homologue, noncoding region etc. of lipid transfer and metabolic gene.

In a further preferred embodiment, the natural antisense sequence of oligonucleotide targeting lipid transfer and metabolic gene target (for coding and noncoding region natural antisense) includes but not limited to variant, allele, homologue, mutant, derivant, fragment and complementary series about it.Preferably, oligonucleotide is antisense RNA or dna molecular.

In a further preferred embodiment, oligomeric compounds of the present invention comprises that also wherein different bases are present in the variant on one or more nucleotide positions in the chemical compound.For example, if first nucleotide is adenine, can be created in so and comprises thymidine, guanosine, cytidine or other variant natural or non-natural nucleotides on this position.This can finish on any position of antisense compounds.These chemical compounds use method described herein to test subsequently, to measure the ability that it suppresses target nucleic acid expression.

In some embodiments, the homology between antisense compounds and target, sequence homogeneity or complementarity are about 50% to about 60%.In some embodiments, homology, sequence homogeneity or complementarity are about 60% to about 70%.In some embodiments, homology, sequence homogeneity or complementarity are about 70% to about 80%.In some embodiments, homology, sequence homogeneity or complementarity are about 80% to about 90%.In some embodiments, homology, sequence homogeneity or complementarity are about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.

The normal function that disturbs target nucleic acid when combining of chemical compound and target nucleic acid is when causing loss of activity, and the complementarity that has enough degree, needing therein avoiding the non-specific binding of antisense compounds and non-target nucleic acid sequence under the bonded condition of specificity, but antisense compounds is a specific hybrid.This type of condition comprise promptly measure in vivo or the therapeutic treatment situation under physiological condition and wherein under the external test situation, carry out the condition of measuring.

The normal function that disturbs target DNA or RNA when combining of chemical compound and target DNA or RNA molecule is when causing the effectiveness forfeiture, and the complementarity that has enough degree, needing therein to avoid the non-specific binding of antisense compounds and non-target nucleic acid sequence under the bonded condition of specificity, promptly measure in vivo or the therapeutic treatment situation under under physiological condition, with under the condition of carry out measuring therein under the external test situation, but antisense compounds no matter be DNA, RNA, chimeric, metathetical etc. be specific hybrid.

In a further preferred embodiment, the antisense sequences that the targeting of lipid transfer and metabolic gene includes but not limited to use for example PCR, hybridization etc. to identify and increase, sequence is one or more etc. shown in SEQ ID NO:8-22, the expression or the function of regulating lipid transfer and metabolic gene.In one embodiment, and compare, express or function raises.In a further preferred embodiment, and compare, express or function is reduced.

In a further preferred embodiment, oligonucleotide comprises the nucleotide sequence shown in SEQ ID NOS:23-263, comprises the antisense sequences that uses for example PCR, hybridization etc. to identify and increase.These oligonucleotide can comprise the nucleotide of one or more modifications, short or than long segment, modifier keys etc.The example of modifier keys or internucleotide linkage comprises thiophosphate, phosphorodithioate etc.In a further preferred embodiment, nucleotide comprises phosphorus derivant.The phosphorus derivant (or modifying phosphate) that can partly adhere to sugar or sugar analogue in modified oligonucleotide of the present invention can be phosplate, bisphosphate, triguaiacyl phosphate, alkylphosphonate, phosphoric acid alkane ester, thiophosphate etc.The preparation of above-mentioned phosphate ester analog, and to mix in the nucleotide of nucleotide, modification and the oligonucleotide itself also be known, and need not to describe in this article.

The specificity and the sensitivity of antisense also are used for the treatment of purposes by those skilled in the art.Antisense oligonucleotide is used as the treatment part in the treatment of the morbid state in the animal and human.Antisense oligonucleotide is applied to the people safely and effectively, and numerous clinical trial is underway at present.Therefore determine that oligonucleotide can be useful treatment pattern, it can be configured in that to be used for the treatment of cell, tissue and animal particularly useful in people's the therapeutic scheme.

In embodiments of the invention, oligomerization antisense compounds particularly oligonucleotide combines with target nucleic acid molecule, and regulates expression and/or function by the molecule of target gene coding.Treat that interferential DNA function comprises and for example duplicate and transcribe.Treat interferential RNA function comprise all great functions for example the RNA transposition to the protein translation site, by RNA translated protein, RNA montage catalytic activity to obtain one or more mRNA kinds and can be connected with RNA or promote by RNA.Depend on required function, this type of function can be to raise or inhibition.

Antisense compounds comprise antisense oligomeric compounds, antisense oligonucleotide, external guide sequence (EGS) oligonucleotide, alternative splicing thing, primer, probe and with other oligomeric compounds of target nucleic acid to small part hybridization.Like this, these chemical compounds can be introduced with the form of strand, two strands, part strand or cyclic oligomeric chemical compound.

In background of the present invention, making antisense compounds targeting specific nucleic acid molecule can be multistep process.This process begins with the evaluation of its function target nucleic acid to be regulated usually.This target nucleic acid can be the cytogene mRNA of genetic transcription (or by) for example, and its expression is relevant with particular disorder or morbid state, or from the nucleic acid molecules of infectious agent.In the present invention, target nucleic acid coding lipid transfer and metabolic gene.

The targeting process also comprises usually being determined at and is used at least one target region, section or the site that antisense interact to take place in the target nucleic acid, thereby makes and will produce required effect, for example adjusting of Biao Daing.In background of the present invention, term " zone " is defined as having the part that at least one can identify the target nucleic acid of structure, function or feature.In the zone of target nucleic acid, be section." section " is defined in the less or inferior subregion in the target nucleic acid.As using in the present invention, " site " is defined in the position in the target nucleic acid.

In a preferred embodiment, antisense oligonucleotide combines with the natural antisense sequence of lipid transfer and metabolic gene, and regulates the expression and/or the function of lipid transfer and metabolic gene (SEQ ID NO:1).The example of antisense sequences comprises SEQ ID NOS:8-263.

In a further preferred embodiment, antisense oligonucleotide combines with one or more sections of lipid transfer and metabolic gene polynucleotide, and regulates the expression and/or the function of lipid transfer and metabolic gene.Section comprises that lipid transfer and metabolic gene have at least 5 continuous nucleotides of justice or antisense polynucleotides.

In a further preferred embodiment, antisense oligonucleotide has specificity for the natural antisense sequence of lipid transfer and metabolic gene, and wherein the natural antisense sequence of oligonucleotide and lipid transfer and metabolic gene combines the expression and/or the function of regulating lipid transfer and metabolic gene.

In a further preferred embodiment, the oligonucleotide chemical compound comprises sequence shown in SEQ ID NOS:23-263, the antisense sequences that uses for example PCR, hybridization etc. to identify and increase.These oligonucleotide can comprise the nucleotide of one or more modifications, short or than long segment, modifier keys etc.The key of modifying or the example of internucleotide linkage comprise thiophosphate, phosphorodithioate etc.In a further preferred embodiment, nucleotide comprises phosphorus derivant.The phosphorus derivant (or phosphate of modifying) that can partly adhere to sugar or sugar analogue in modified oligonucleotide of the present invention can be phosplate, bisphosphate, triguaiacyl phosphate, alkylphosphonate, phosphoric acid alkane ester, thiophosphate etc.The preparation of above-mentioned phosphate ester analog, and to mix in the nucleotide of nucleotide, modification and the oligonucleotide itself also be known, and need not to describe in this article.

Because as known in the art, translation initiation codon generally is that 5'-AUG(is in the mRNA molecule of transcribing; Be 5'-ATG in corresponding dna molecular), so translation initiation codon is also referred to as " AUG codon ", " start codon " or " AUG start codon ".The minority gene has the translation initiation codon that contains RNA sequence 5'-GUG, 5'-UUG or 5'-CUG; And 5'-AUA, 5'-ACG and 5'-CUG have shown in vivo and have worked.Therefore, term " translation initiation codon " and " start codon " can comprise many codon sequences, even the initial sub-aminoacid under every kind of situation generally is methionine (in eukaryote) or formylmethionine (in prokaryote).Eukaryote and prokaryotic gene can have 2 or more a plurality of variable start codon, and wherein any one can preferentially be used at particular cell types or tissue or the translation initiation under the specified conditions group.In background of the present invention, " start codon " and " translation initiation codon " refers to so one or more codons, it is used for the translation of the mRNA of initial genetic transcription by coding lipid transfer and metabolic gene in vivo, and is irrelevant with one or more sequences of this type of codon.One of it is that the corresponding DNA sequence with 5'-UGA(of 5'-UAA, 5'-UAG is respectively 5'-TAA, 5'-TAG and 5'-TGA that translation stop codon of gene (or " termination codon ") can have three kinds of sequences).

Term " initiation codon subarea " and " translation initiation codon district " refer to the part of this type of mRNA or gene, its comprise from translation initiation codon begin either direction (that is, 5' or 3') about 25 to about 50 in abutting connection with nucleotide.Similarly, term " termination codon subarea " and " translation termination codon region " refer to the part of this type of mRNA or gene, its comprise from translation stop codon beginning either direction (that is, 5' or 3') about 25 to about 50 in abutting connection with nucleotide.Therefore, " initiation codon subarea " (or " translation initiation codon district ") and " termination codon subarea " (or " translation termination codon region ") are can be with the All Ranges of antisense compounds efficient targeting of the present invention.

Open reading-frame (ORF) known in the art, as to refer to the zone between translation initiation codon and translation stop codon or " coding region " also be can efficient targeting the zone.In background of the present invention, target area is to comprise the translation initiation of gene open reading-frame (ORF) or the territory, intron of termination codon.

Another kind of target region comprises 5' untranslated region known in the art (5'UTR), finger begins mRNA part the 5' direction from translation initiation codon, and therefore is included in 5' cap site and the nucleotide between the translation initiation codon (or the corresponding nucleotide on the gene) of mRNA.Another target region comprises 3' untranslated region known in the art (3'UTR), finger is from the mRNA part of translation stop codon beginning the 3' direction, and therefore is included in translation stop codon and the nucleotide between the 3' end (or the corresponding nucleotide on the gene) of mRNA.The 5' cap site of mRNA comprises the guanosine residue that methylates of the N7-via the 5' least significant end residue adjacency of 5'-5' triphosphoric acid key and mRNA.The 5' of mRNA adds the medicated cap district and is regarded as comprising that 5' adds cap self and preceding 50 nucleotide adjacent with cap site.About another kind of target region of the present invention is that 5' adds the medicated cap district.

Although some eukaryote mRNA transcripies are directly translations, many one or more zones that are called " intron " that comprise, this excises from transcript before its translation.All the other (with therefore translation) zones are called " exon ", and together montage to form continuous mRNA sequence.In one embodiment, the targeting splice site is that intron-exon connects or exon-intron connection is useful especially in the following cases, when aberrant splicing involves disease, or when the overproduction of specific montage product involves disease.Because it is another embodiment of target site that the unusual fusion of resetting or lacking connects.The mRNA transcript that produces via the montage process from 2 kinds of (or more kinds of) mRNAs in different genes source is called " fusion transcript ".Intron can use for example antisense compounds efficient targeting of DNA or mRNA precursor of targeting.

In a further preferred embodiment, antisense oligonucleotide combines with the coding and/or the noncoding region of target polynucleotide, and regulates the expression and/or the function of target molecule.

In a further preferred embodiment, antisense oligonucleotide combines with the natural antisense polynucleotide, and regulates the expression and/or the function of target molecule.

In a further preferred embodiment, antisense oligonucleotide combines with adopted polynucleotide are arranged, and regulates the expression and/or the function of target molecule.

The altered rna transcript can be produced by the homologous genes group zone of DNA.These alternative transcription things are commonly referred to as " variant ".More specifically, " mRNA precursor variant " is the transcript that is produced by homologous genes group DNA, and it is different from other transcripies that produced by homologous genes group DNA at it in the initial or final position, and comprises intron and exon sequence.

After one or more exons or intron zone or the excision of its part, mRNA precursor variant produces littler " mRNA variant " in the montage process.Therefore, the mRNA variant is the mRNA precursor variant through processing, and because montage, every kind of unique mRNA precursor variant must produce unique mRNA variant always.These mRNA variants are also referred to as " alternative splicing variant ".If the montage of mRNA precursor variant does not take place, mRNA precursor variant is equal to the mRNA variant so.

Variant can be transcribed by using variable signal to produce with initial or termination.MRNAs precursor and mRNAs can have more than a start codon or termination codon.The variant that comes from the mRNA precursor that uses variable start codon or mRNA is called " the variable initial variant " of the sort of mRNA precursor or mRNA.Use these transcripies of variable termination codon to be called the sort of mRNA precursor or mRNA " variable termination variant ".The variable termination variant of a particular type is " a polyadenylic acid variant ", the a plurality of transcripies that wherein produced result from by the variable selection of one of " polyadenylic acid termination signal " of the mechanism of transcribing, thereby are created in the transcript that stops on unique polyadenylic acid site.In background of the present invention, variant type described herein also is the embodiment of target nucleic acid.

Location on the target nucleic acid that antisense compounds is hybridized with it is defined as its long part of the nucleotide of 5-at least of target region of active antisense compounds targeting.

Although set forth the concrete sequence of particular exemplary target area section in this article, those skilled in the art will recognize that these are used for illustrating and describe particular within the scope of the present invention.The target area section can consider that present disclosure easily identifies by those of ordinary skills in addition.

The target area section that comprises length 5-100 the nucleotide that is selected from least five (5) individual continuous nucleotide sections in the section of the preferred target area of illustrative is regarded as also being suitable for targeting.

The target area section can comprise DNA or RNA sequence, and it comprises from least 5 continuous nucleotides of the terminal beginning of 5' of one of the preferred target area of illustrative section (all the other nucleotide are tightly to begin and continue to comprise about 5 same DNA or RNA continuous segments to about 100 nucleotide until DNA or RNA in the terminal upstream of the 5' of target area section).Similar preferred target area section is represented that by DNA or RNA sequence it comprises from least 5 continuous nucleotides of the terminal beginning of 3' of one of the preferred target area of illustrative section (all the other nucleotide are tightly to begin and continue to comprise about 5 same DNA or RNA continuous segments to about 100 nucleotide until DNA or RNA in the terminal downstream of the 3' of target area section).Need not undo experimentation, the those skilled in the art that prepare with the illustrational target area of this paper section can identify further preferred target area section.

In case identified one or more target regions, section or site, just select and the abundant complementary antisense compounds of target, promptly fully good and have enough specific hybrids, to provide required effect.

In one embodiment of the invention, oligonucleotide combines with the antisense strand of particular target.Oligonucleotide length is at least 5 nucleotide, and can so synthesize each oligonucleotide target overlapping sequence, thereby makes the synthetic oligonucleotide that covers the complete length of target polynucleotide.Target also comprises coding and noncoding region.

In one embodiment, preferably by antisense oligonucleotide targeting specific nucleic acid.Making antisense compounds targeting specific nucleic acid is multistep process.This process begins with the evaluation of its function nucleotide sequence to be regulated usually.This can be for example to express the cytogene relevant with particular disorder or the morbid state mRNA of genetic transcription (or by), or non-coded polynucleotide non-coding RNA (ncRNA) for example.

RNAs can be categorized into (1) and translate into proteinic messenger RNA s(mRNAs) and (2) nonprotein coding RNA s(ncRNAs).NcRNAs comprises Microrna s, antisense transcript and comprises high density termination codon and lack any extensively other transcript units (TU) of " open reading-frame ".Many ncRNAs seem that the initiation site from the 3' untranslated region (3'UTRs) of protein coding gene seat begins.NcRNAs is normally rare, and half ncRNAs at least by FANTOM association order-checking seems not to be polyadenylic acidization.Owing to conspicuous reason, most researchers concentrates on processed and outputs to cytoplasmic polyadenylic acid mRNAs.Recently, the nRNA s group that has shown non-polyadenylic acidization can be greatly, and many this type of transcripies arise from so-called intergenic region (Cheng, people such as J. (2005). Science308(5725), 1149-1154; Kapranov, people such as P. (2005). Genome Res 15 (7), 987-997)。The mechanism of ncRNAs by its scalable gene expression be by with the base pairing of target transcript.The RNAs that works by base pairing can be grouped into (1) cis coding RNA s, it is coding on the homologous genes location, but on opposite strand at RNAs, they act on and therefore show the perfect complementarity with its target, (2) RNAs of trans coding, it is encoded acting on the different chromosome mapping of its RNAs with them, and does not generally demonstrate the perfect base pairing potentiality with its target.

Be not wishing to be bound by theory, disturb antisense polynucleotides, can change the expression that adopted messenger RNA s should be arranged mutually by antisense oligonucleotide described herein.Yet this adjusting can be (antisense is knocked down the messenger RNA that causes following and reduced) of inharmonious (antisense is knocked down and caused messenger RNA to raise) or coordination.In these cases, the overlapping or non-overlapped part that antisense oligonucleotide can targeting antisense transcript causes it to knock down or isolates (sequestration).Coding and non-encoding antisense can be with the equivalent way targeting, and arbitrary category can both be regulated and should have justice to transcribe thing – to coordinate or inharmonious mode mutually.Being used for the strategy that adopts at the new oligonucleotide that target uses in evaluation can be based on the knocking down of antisense RNA transcript, and it is by antisense oligonucleotide or any other mode of regulating required target.

Strategy 1: under not coordinately regulated situation, knock down conventional (justice is arranged) expression of gene of antisense transcript rising.A kind of gene in back should be encoded known or supposition medicine target, then the effect of knocking down simulated receptor agonist conceivably or enzyme stimulus object of its antisense counter pair.

Strategy 2: under coordinately regulated situation, thereby can knock down antisense concomitantly and adopted transcript is arranged and reach the collaborative reduction of conventional (justice is arranged) gene expression.If for example antisense oligonucleotide is used to reach and knocks down, so this strategy can be used to use targeting at a kind of antisense oligonucleotide that adopted transcript is arranged, with another kind of antisense oligonucleotide for corresponding antisense transcript, or the overlapping single effectively symmetric antisense oligonucleotide that justice and antisense transcript are arranged of while targeting.

According to the present invention, antisense compounds comprise antisense oligonucleotide, ribozyme, external guide sequence (EGS) oligonucleotide, siRNA chemical compound, list or double-stranded RNA disturb (RNAi) chemical compound for example the siRNA chemical compound and with target nucleic acid to small part hybridization and regulate other oligomeric compounds of its function.Like this, they can be DNA, RNA, DNA sample, RNA sample or its mixture, maybe can be one or more the analogies in these.These chemical compounds can be strand, two strands, ring-type or hair clip oligomeric compounds, and can comprise structural detail for example inside or end-boss, mispairing or ring.The linear preparation of antisense compounds more solito, but can connect or otherwise preparation, to be ring-type and/or ramose.Antisense compounds can comprise 2 chains that construct is for example hybridized, and with the double-stranded wholly or in part chemical compound of formation, or has enough complementary strands of oneself, to allow to hybridize and form the chemical compound of all or part of two strands.Article 2, chain can innerly connect, and stays free 3' or 5' end, maybe can connect to form continuous hairpin structure or ring.Hairpin structure can be included in the jag on 5' or the 3' end, produces the prolongation of strand feature.The optional jag that can be included on the end of double chain compound.Further modification can comprise the conjugate group that one of bonding adheres between nucleotide position, sugared position or nucleoside with one of terminal, selection.Alternately, 2 chains can connect via non-nucleic acid moiety or joint group.When by 1 chain formation only, dsRNA can take self complementary hair clip type molecular forms, and it turns back on himself to form duplex.Therefore, dsRNAs can be all or part of two strands.The specificity of gene expression is regulated and can be reached by the stably express of dsRNA hair clip in transgenic cell line, yet in some embodiments, gene expression or function raise.When being formed by 2 chains or strand, described strand is taked to turn back on himself to form self complementary hair clip type molecular forms of duplex, and 2 chains (or the duplex of strand forms the zone) are the complementary RNA chains with Wo Sen-Ke Like form base pairing.

In case in the drawing-in system, chemical compound of the present invention just can cause the effect of one or more enzymes or structural protein, realizing cutting or other modifications of target nucleic acid, or can be via based on occupying mechanism works.Generally speaking, nucleic acid (comprising oligonucleotide) can be described as " the DNA sample " (promptly, generally have one or more 2'-deoxysaccharides, and generally be T rather than U base) or " RNA sample " is (promptly, generally have the sugar that one or more 2'-hydroxyls or 2'-modify, and generally be U rather than T base).The nucleic acid spiral can adopt and surpass one type structure, the most common A and B shape.Generally speaking, think that the oligonucleotide with B shape spline structure is " a DNA sample ", and the oligonucleotide with A shape spline structure is " a RNA sample ".In some (chimeric) embodiments, antisense compounds can comprise A and B shape zone.

In a further preferred embodiment, required oligonucleotide or antisense compounds comprise following at least a: antisense RNA, antisense DNA, chimeric antisense oligonucleotide, the antisense oligonucleotide that comprises the bonding of modification, RNA interfering (RNAi), short interfering rna (siRNA); Small, RNA interfering (miRNA); Little, instantaneous RNA(stRNA); Or short, hairpin RNA (shRNA); The inductive gene activation of little RNA (RNAa); Little activator RNA s(saRNAs) or its combination.

DsRNA can also activated gene expresses, and this is the mechanism of called after " the inductive gene activation of little RNA " or RNAa.The dsRNAs of target gene promoter induces effective transcriptional activation of related gene.RNAa uses synthetic dsRNAs to confirm that in people's cell called after " little activator RNA s " (saRNAs).Whether present unknown RNAa guards in other biological.

Little double-stranded RNA (dsRNA) for example siRNA (siRNA) or Microrna (miRNA) has found it is to be called the triggering thing that RNA disturbs the evolution conservative mechanism of (RNAi).RNAi always causes via reinventing chromatinic gene silencing, thereby transcribes, degrades the translation of complementary mRNA or blocking protein to check.Yet under the situation about describing in detail in the embodiment sections, oligonucleotide shows expression and/or the function that increases lipid transfer and metabolic gene polynucleotide and coded product thereof hereinafter.DsRNAs can also serve as little activator RNA s(saRNA).Be not wishing to be bound by theory, by the sequence in the target gene promoter, saRNAs will induce expression of target gene in the phenomenon that is called as dsRNA inducible transcription activation (RNAa).

In a further embodiment, " preferred target area section " that this paper identifies can be used to screen the other chemical compound of regulating lipid transfer and the expression of metabolic gene polynucleotide." regulator " is such chemical compound, and the nucleic acid molecules of its minimizing or increase coding lipid transfer and metabolic gene is expressed, and comprises and the preferred complementary 5-at least of target area section nucleotide segment.Screening technique comprises step: the preferred target area section of the nucleic acid molecules that justice or natural antisense polynucleotide are arranged of coding lipid transfer and metabolic gene is contacted with one or more candidate modulator, and one or more candidate modulator that the nucleic acid molecules of selecting minimizing or increase coding lipid transfer and metabolic gene polynucleotide is expressed, described nucleic acid molecules is SEQ ID NO:23-263 for example.In case showing one or more candidate modulator can regulate the nucleic acid molecules of (for example reduce or increase) coding lipid transfer and metabolic gene polynucleotide and express, regulator just can be used for the further investigation of lipid transfer and metabolic gene polynucleotide function subsequently, or is used for being used as according to research of the present invention, diagnosis or therapeutic agent.

Targeting natural antisense sequence preference is regulated the function of target gene.For example, and lipid transfer and metabolic gene gene (for example registration number NM_005502, NM_000229, NM_002332, NM_008512, NM_000527.3, NM_000041, NM_000039, Fig. 2).In a preferred embodiment, target is the antisense polynucleotides of lipid transfer and metabolic gene.In a preferred embodiment, antisense oligonucleotide targeting lipid transfer and metabolic gene polynucleotide are (for example, registration number NM_005502, NM_000229, NM_002332, NM_008512, NM_000527, NM_000041, NM_000039, Fig. 2) justice and/or natural antisense sequence arranged, about its variant, allele, isotype, homologue, mutant, derivant, fragment and complementary series.Preferably, oligonucleotide is an antisense molecule, and target comprises antisense and/or adopted lipid transfer arranged and the coding and the noncoding region of metabolic gene polynucleotide.

Preferred target area of the present invention section can also make up with other complementary antisense compounds of the present invention of its branch, to form stable double-stranded (duplex) oligonucleotide.

This type of double chain oligonucleotide part has shown that in the art regulating target expresses, and regulates translation and RNA processing via antisense mechanism.In addition, can partly implement chemical modification (people such as Fire, (1998) to two strands Nature, 391,806-811; Timmons and Fire, (1998) Nature, 395,854; People such as Timmons, (2001) Gene, 263,103-112; People such as Tabara, (1998) Science, 282,430-431; People such as Montgomery (1998), Proc. Natl. Acad. Sci. USA, 95,15502-15507; People such as Tuschl, (1999) Genes Dev., 13,3191-3197; People such as Elbashir, (2001) Nature, 411,494-498; People such as Elbashir, (2001) Genes Dev., 15,188-200).For example, this type of double-stranded part has shown by the antisense strand of duplex and the typical case of target hybridizes the inhibition target, thus the enzymatic degradation of triggering target (people such as Tijsterman, (2002) Science, 295,694-697).

In a preferred embodiment, antisense oligonucleotide targeting lipid transfer and metabolic gene polynucleotide (for example, registration number NM_005502, NM_000229, NM_002332, NM_008512, NM_000527, NM_000041, NM_000039), about its variant, allele, isotype, homologue, mutant, derivant, fragment and complementary series.Preferably, oligonucleotide is an antisense molecule.

According to embodiment of the present invention, target nucleic acid molecule is not limited to independent lipid transfer and metabolic gene, also extends to any in the isotype, receptor, homologue etc. of lipid transfer and metabolic gene molecule.

In a further preferred embodiment, the natural antisense sequence of oligonucleotide targeting lipid transfer and metabolic gene polynucleotide, polynucleotide shown in SEQ ID NO:8-22 for example, and about its any variant, allele, homologue, mutant, derivant, fragment and complementary series.The example of antisense oligonucleotide is shown in SEQ ID NO:23-263.

In one embodiment, the nucleic acid array complementation of oligonucleotide and lipid transfer and metabolic gene antisense or combine, include but not limited to that the non-coding relevant with the metabolic gene polynucleotide with lipid transfer has justice and/or antisense sequences, and regulate the expression and/or the function of lipid transfer and metabolic gene molecule.

In a further preferred embodiment, the nucleic acid array complementation of oligonucleotide and lipid transfer shown in SEQ ID NO:8-22 and metabolic gene natural antisense or combine, and regulate the expression and/or the function of lipid transfer and metabolic gene molecule.

In a preferred embodiment, oligonucleotide comprises the sequence of at least 5 continuous nucleotides of SEQ ID NOS:23-263, and regulates the expression and/or the function of lipid transfer and metabolic gene molecule.

The polynucleotide target comprises lipid transfer and metabolic gene, comprises the variant of its family member, lipid transfer and metabolic gene; The mutant of lipid transfer and metabolic gene comprises SNPs; The non-coding sequence of lipid transfer and metabolic gene; The allele of lipid transfer and metabolic gene; Species variant, fragment etc.Preferably, oligonucleotide is an antisense molecule.

In a further preferred embodiment, the oligonucleotide of targeting lipid transfer and metabolic gene polynucleotide comprises: antisense RNA, RNA interfering (RNAi), short interfering rna (siRNA); Subtle disruption RNA(miRNA); Little, instantaneous RNA(stRNA); Or short, hairpin RNA (shRNA); The inductive gene activation of little RNA (RNAa); Or little activator RNA (saRNA).

In a further preferred embodiment, lipid transfer and the metabolic gene polynucleotide targeting of SEQ ID NO:8-22 expression or the function of regulating these targets for example.In one embodiment, and compare, express or function raises.In a further preferred embodiment, and compare, express or function is reduced.

In a further preferred embodiment, antisense compounds comprises sequence shown in SEQ ID NOS:23 – 263.These oligonucleotide can comprise the nucleotide of one or more modifications, short or than the key of long segment, modification etc.

In a further preferred embodiment, SEQ ID NOS:23 – 263 comprises one or more LNA nucleotide.

The adjusting of required target nucleic acid can be carried out with several means known in the art.For example, antisense oligonucleotide, siRNA etc.Enzymatic nucleic acid molecules (for example, ribozyme) is one or more the nucleic acid molecules in can the various reactions of catalysis, comprises in nucleotide base sequence specificity mode and repeats to cut the ability that other separate nucleic acid molecules.This type of enzymatic nucleic acid molecules can for example be used for the in fact any rna transcription thing of targeting (people such as Zaug, 324, Nature429 1986; Cech, 260 JAMA3030,1988; With people such as Jefferies, 17 Nucleic Acids Research1371,1989).

Because its sequence-specific, so the enzymatic nucleic acid molecules of trans cutting shows as the hope (Usman and McSwiggen, (1995) that are used for human disease's therapeutic agent Ann. Rep. Med. Chem. 30,285-294; Christoffersen and Marr, (1995) J. Med. Chem. 38,2023-2037).The enzymatic nucleic acid molecules can be designed as the specific RNA target of cutting in the cell RNA background.This type of cutting incident causes mRNA not have function and cancellation from the protein expression of the sort of RNA.By this way, can suppress the protein synthesis relevant by selectivity with morbid state.

Generally speaking, the enzymatic nucleic acid with RNA cleavage activity works by at first combining with target RNA.This type of takes place in conjunction with the target bound fraction by enzymatic nucleic acid, and the enzymatic of described enzymatic nucleic acid and molecule partly keeps closely approaching, and described molecular action is in cutting target RNA.Therefore, enzymatic nucleic acid at first discern and subsequently by complementary base pairing in conjunction with target RNA, and in case combine with correct site, with regard to enzymatic catalysis with cutting target RNA.The tactic cutting of this type of target RNA will destroy it and instruct the synthetic ability of coded protein.Enzymatic nucleic acid in conjunction with and cut its RNA target after, it discharges from the sort of RNA searching for another target, and can repeat combination and cut new target.

The for example external selection of several method (evolution) strategy (Orgel, (1979) Proc. R. Soc. London, B 205,435) be used to evolve can the various reactions of catalysis the novel nucleic acids catalyst, for example the cutting of phosphodiester bond and amido link be connected (Joyce, (1989) Gene, 82,83-87; People such as Beaudry, (1992) Science257,635-641; Joyce, (1992) Scientific American 267,90-97; People such as Breaker, (1994) TIBTECH12,268; People such as Bartel, (1993) Science261:1411-1418; Szostak, (1993) TIBS17,89-93; People such as Kumar, (1995) FASEB J., 9,1183; Breaker, (1996) Curr. Op. Biotech., 7,442).

To significantly facilitate for the exploitation of the ribozyme of catalytic activity the best and to adopt RNA cutting ribozyme to be used for any strategy that regulator gene is expressed purpose.Hammerhead ribozyme is for example at the Mg of saturated (10 mM) concentration ²⁺Under the existence of cofactor with about 1 minute ^-1Catalytic rate (k _Cat) work.Manually " RNA ligase " ribozyme has shown with about 100 minutes ^-1The corresponding self-modification reaction of speed catalysis.In addition, known hammerhead ribozyme catalysis RNA with specific modification of the substrate brachium conjunctivum of being made by DNA cuts, and has to reach 100 minutes ^-1Multiple turnover rate.At last, replacing specific residue in the catalytic core of tup with the specific nucleotide analog is given in and shows the nearly modification ribozyme of 10 times of improvement in the catalytic rate.These find to confirm that ribozymes can promote chemical conversion, its catalytic rate obviously greater than by most of natural self-cutting ribozymes at external those that show.Structure that subsequently may can the specific self-cutting ribozyme of optimization to provide catalytic activity to greatest extent, maybe can prepare new RNA motif fully, and it shows for the RNA di-phosphate ester and cuts obvious faster rate.

The RNA substrate at first showed (Uhlenbeck, O. C.(1987) by the intermolecular cutting of the RNA catalyst of match " tup " model in 1987 Nature, 328:596-600).The RNA catalyst recover and with multiple RNA molecular reaction, confirm that it is real catalytic.

Catalysis RNAs based on " tup " motif design has been used for the cleavage specificity target sequence, and this is by carrying out suitable sequence change in catalysis RNA, to keep essential base pairing (Haseloff and Gerlach, (1988) with target sequence NaturE, 334,585; Walbot and Bruening, (1988) Nature, 334,196; Uhlenbeck, O. C.(1987) Nature, 328:596-600; Koizumi, M. waits people (1988) FEBS Lett., 228:228-230).This has allowed to use catalysis RNA with the cleavage specificity target sequence, and point out according to the catalysis RNA of " tup " modelling can be in vivo cleavage specificity substrate RNA s possibly.(referring to Haseloff and Gerlach, (1988) Nature, 334,585; Walbot and Bruening, (1988) Nature, 334,196; Uhlenbeck, O. C.(1987) Nature, 328:596-600).

RNA disturbs (RNAi) to become to be used for the strong instrument of the gene expression of regulating mammal and mammalian cell.This method requires to send as RNA self or as the siRNA (siRNA) of DNA, and it uses expression plasmid or virus and is used to be processed as the coded sequence of the bobby pin RNAs of siRNAs.This system makes the siRNAs precursor can effectively be transported to their active therein Cytoplasms, and allows to use adjusting and tissue-specific promoter to be used for gene expression.

In a preferred embodiment, oligonucleotide or antisense compounds comprise ribonucleic acid (RNA) and/or DNA (deoxyribonucleic acid) (DNA), or the oligomer of its analogies, chimera, analog or homologue or polymer.This term comprises the oligonucleotide of being made up of (main chain) bonding between naturally occurring nucleotide, sugar and covalency nucleoside, and there is oligonucleotide partly in the non-natural that has that works similarly.Usually need this type of modification or the oligonucleotide that replaces surpass native form since required character for example cellular uptake strengthen, strengthen and stability in the presence of nuclease increases for the affinity of target nucleic acid.

According to the present invention, oligonucleotide or " antisense compounds " comprise antisense oligonucleotide (for example, RNA, DNA, its analogies, chimera, analog or homologue), ribozyme, external guide sequence (EGS) oligonucleotide, siRNA chemical compound, list or double-stranded RNA disturb (RNAi) chemical compound for example siRNA chemical compound, saRNA, aRNA and with target nucleic acid to small part hybridization and regulate other oligomeric compounds of its function.Like this, they can be DNA, RNA, DNA sample, RNA sample or its mixture, maybe can be one or more the analogies in these.These chemical compounds can be strand, two strands, ring-type or hair clip oligomeric compounds, and can comprise structural detail for example inside or end-boss, mispairing or ring.The linear preparation of antisense compounds more solito, but can connect or otherwise preparation, to be ring-type and/or ramose.Antisense compounds can comprise 2 chains that construct is for example hybridized, and with the double-stranded wholly or in part chemical compound of formation, or has enough complementary strands of oneself, to allow to hybridize and form the chemical compound of all or part of two strands.Article 2, chain can innerly connect, and stays free 3' or 5' end, maybe can connect to form continuous hairpin structure or ring.Hairpin structure can be included in the jag on 5' or the 3' end, produces the prolongation of strand feature.The optional jag that can be included on the end of double chain compound.Further modification can comprise the conjugate group that one of bonding adheres between nucleotide position, sugared position or nucleoside with one of terminal, selection.Alternately, 2 chains can connect via non-nucleic acid moiety or joint group.When by 1 chain formation only, dsRNA can take self complementary hair clip type molecular forms, and it turns back on himself to form duplex.Therefore, dsRNAs can be all or part of two strands.(people such as Hammond, (1991) are regulated and can be reached by the stably express of dsRNA hair clip in transgenic cell line to the specificity of gene expression Nat. Rev. Genet., 2,110-119; People such as Matzke, (2001) Curr. Opin. Genet. Dev., 11,221-227; Sharp, (2001) Genes Dev., 15,485-490).When being formed by 2 chains or strand, described strand is taked to turn back on himself to form self complementary hair clip type molecular forms of duplex, and 2 chains (or the duplex of strand forms the zone) are the complementary RNA chains with Wo Sen-Ke Like form base pairing.

In case in the drawing-in system, chemical compound of the present invention just can cause the effect of one or more enzymes or structural protein, realizing cutting or other modifications of target nucleic acid, or can be via based on occupying mechanism works.Generally speaking, nucleic acid (comprising oligonucleotide) can be described as " the DNA sample " (promptly, generally have one or more 2'-deoxysaccharides, and generally be T rather than U base) or " RNA sample " is (promptly, generally have the sugar that one or more 2'-hydroxyls or 2'-modify, and generally be U rather than T base).The nucleic acid spiral can adopt and surpass one type structure, the most common A and B shape.Generally speaking, think that the oligonucleotide with B shape spline structure is " a DNA sample ", and the oligonucleotide with A shape spline structure is " a RNA sample ".In some (chimeric) embodiments, antisense compounds can comprise A and B shape zone.

Can comprise about 80 nucleotide of about 5 – of the length antisense part of (about 80 of promptly about 5 – connect nucleoside) according to antisense compounds of the present invention.This is meant the antisense strand of antisense compounds or the length of part.In other words, strand antisense compounds of the present invention comprises about 80 nucleotide of 5 –, and double-stranded antisense compounds of the present invention (for example dsRNA) comprises adopted and the antisense strand or the part of having of about 80 nucleotide of length 5 –.Those of ordinary skills are to be understood that this comprises length 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79 or 80 nucleotide, or the antisense part of any scope in it.

In one embodiment, antisense compounds of the present invention has the antisense part of 50 nucleotide of length 10 –.Those of ordinary skills are to be understood that this has embodied and has 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 or 50 nucleotide of length, or the oligonucleotide of the antisense of any scope in it part.In some embodiments, oligonucleotide length is 15 nucleotide.

In one embodiment, antisense of the present invention or oligonucleotide chemical compound have the antisense part of length 12 or 30 nucleotide of 13 –.Those of ordinary skills are to be understood that this has embodied and has 12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 nucleotide of length, or the antisense compounds of the antisense of any scope in it part.

In a further preferred embodiment, oligomeric compounds of the present invention comprises that also wherein different bases are present in the variant on one or more nucleotide positions in the chemical compound.For example, if first nucleotide is adenosine, can be created in the variant that comprises thymidine, guanosine or cytidine on this position so.This can finish on any position of antisense or dsRNA chemical compound.These chemical compounds use method described herein to test subsequently, to measure the ability that it suppresses target nucleic acid expression.

In some embodiments, the homology between antisense compounds and target, sequence homogeneity or complementarity are about 40% to about 60%.In some embodiments, homology, sequence homogeneity or complementarity are about 60% to about 70%.In some embodiments, homology, sequence homogeneity or complementarity are about 70% to about 80%.In some embodiments, homology, sequence homogeneity or complementarity are about 80% to about 90%.In some embodiments, homology, sequence homogeneity or complementarity are about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.

In a further preferred embodiment, antisense oligonucleotide for example the nucleic acid molecules shown in the SEQ ID NO:8-263 comprise one or more displacements or modification.In one embodiment, nucleotide is by lock nucleic acid (LNA) displacement.

In a further preferred embodiment, the nucleic acid molecules of oligonucleotide targeting and lipid transfer and metabolic gene and sequence is relevant shown in SEQ ID NO:1-7 and 8-22 coding and/or non-coding sequence has one or more zones of justice and/or antisense.Oligonucleotide is the overlapping region of targeting SEQ ID NO:1-7 and 8-22 also.

Certain preferred oligonucleotide of the present invention is a chimeric oligonucleotide." chimeric oligonucleotide " in background of the present invention or " chimera " are the oligonucleotide that comprises 2 or more a plurality of chemically zoness of different, and each free at least one nucleotide constitutes.These oligonucleotide generally comprise (for example gives one or more favourable character, the nuclease resistance increases, enters intracellular picked-up and increases, increases for the binding affinity of target) at least one zone of nucleotide of modification and its be zone about the substrate of the enzyme that can cut RNA:DNA or RNA:RNA hybrid.For example, RNA enzyme H is a cellular endonuclease, the RNA chain of its cutting RNA:DNA duplex.Therefore the activation of RNA enzyme H causes the cutting of RNA target, thereby greatly the antisense of reinforcing gene expression is regulated efficient.Therefore, when using chimeric oligonucleotide, with the thiophosphate deoxy-oligonucleotide of identical target region hybridization relatively, use than short oligonucleotide and can obtain suitable with it result usually.The cutting of RNA target can more solito by gel electrophoresis and when needing associated nucleic acid hybridization technology known in the art detect.In a preferred embodiment, chimeric oligonucleotide comprises and is modified at least one zone that increases the target binding affinity and serves as zone about the substrate of RNA enzyme H usually.Oligonucleotide passes through to measure the right T of oligonucleotide/target for the affinity of its target (nucleic acid of coding ras in this case) _mMore solito is measured, T _mBe oligonucleotide and the dissociated temperature of target under it; Metric measurement ground detects and dissociates.T _mHigh more, oligonucleotide is big more for the affinity of target.

The composite construction that chimeric antisense compounds of the present invention can be used as aforesaid two or more oligonucleotide, modified oligonucleotide, oligonucleoside and/or oligonucleotide mimetic forms.This compounds has been called as hybrid or gapmer in the art.Instruct the representative United States Patent (USP) of this type of hybrid structure preparation to include but not limited to U.S. Patent number 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356 and 5,700,922; It is incorporated herein by reference separately.

In a further preferred embodiment, the oligonucleotide zone of modification is included at least one nucleotide of modifying on the 2' position of sugar, most preferably the nucleotide of 2'-O-alkyl, 2'-O-alkyl-O-alkyl or the modification of 2'-fluoro-.In other preferred embodiments, RNA modifies the 2'-fluorine on the ribose of pyrimidine, dealkalize base residue or reversing base, 2'-amino and the modification of 2'O-methyl on the 3' end that is included in RNA.This type of is modified more solito and mixes in the oligonucleotide, and these oligonucleotide have shown to have and are higher than the T of 2'-deoxy-oligonucleotide at given target _m(that is, higher target binding affinity).The effect of the affinity of this type of increase is for the greatly RNAi oligonucleotide inhibition of reinforcing gene expression.RNA enzyme H is the cellular endonuclease of the RNA chain of cutting RNA:DNA duplex; Therefore the activation of this kind of enzyme causes the cutting of RNA target, and therefore can greatly strengthen the efficient that RNAi suppresses.The cutting of RNA target can more solito be confirmed by gel electrophoresis.In a further preferred embodiment, chimeric oligonucleotide is also modified, to strengthen the nuclease resistance.Cell comprises the circumscribed and restriction endonuclease of various nucleic acid of the nucleic acid of can degrading.Many nucleotide and nucleoside modification have shown makes the oligonucleotide that they are introduced wherein to nuclease degradation resistance be arranged more than natural oligodeoxynucleotide.Nuclease resistance more solito is measured: hatch with cell extract or isolating nucleic acid enzymatic solution by making oligonucleotide, and measure along with remaining in the past complete oligonucleotide degree of time by gel electrophoresis usually.The oligonucleotide of having modified to strengthen its nuclease resistance is kept perfectly the longer time than unmodified oligonucleotide.The various oligonucleotides-modified nuclease resistances that confirmed to strengthen or give.The oligonucleotide that comprises at least a thiophosphate modification is preferred at present.In some cases, the oligonucleotides-modified of intensifier target binding affinity also can strengthen the nuclease resistance independently.Some wish to modify can be people (1995) such as De Mesmaeker Acc. Chem. Res., find among the 28:366-374.

The object lesson that imagination is used for preferred oligonucleotide more of the present invention comprises and comprises those that modify main chain, for example bonding between bonding or short chain hetero atom or heterocycle sugar between thiophosphate, phosphotriester, methyl phosphonate, short-chain alkyl or cycloalkyl sugar.Most preferably have the oligonucleotide of thiophosphate main chain and have those of hetero atom main chain, particularly CH ₂--NH--O--CH ₂, CH,--N(CH ₃)--O--CH ₂[being called methylene (methyl-imino) or MMI main chain], CH ₂--O--N(CH ₃)--CH ₂, CH ₂--N(CH ₃)--N(CH ₃)--CH ₂And O--N(CH ₃)--CH ₂--CH ₂Main chain, wherein the natural phosphodiester main chain is expressed as O--P--O--CH).By people (1995) such as De Mesmaeker Acc. Chem. Res., the disclosed amide backbone of 28:366-374 also is preferred.The oligonucleotide (Summerton and Weller, U.S. Patent number 5,034,506) that further preferably has the morpholino backbone structure.In other preferred embodiments, peptide nucleic acid(PNA) (PNA) main chain for example, the phosphodiester backbone of oligonucleotide is replaced by polyamide skeleton, and nucleotide directly or indirectly combines (people (1991) such as Nielsen with the aza nitrogen atom of polyamide skeleton Science, 254,1497).Oligonucleotide can also comprise one or more replacement sugar moieties.Preferred oligonucleotide comprises one of the following: OH, SH, SCH on the 2' position ₃, F, OCN, OCH ₃OCH ₃, OCH ₃O(CH ₂) _nCH ₃, O(CH ₂) _nNH ₂Or O(CH ₂) _nCH ₃, wherein n is 1 – about 10; C ₁-C ₁₀Low alkyl group, alkaryl or the aralkyl of low alkyl group, alkoxyl alkoxyl, replacement; Cl; Br; CN; CF ₃OCF ₃O--, S--or N-alkyl; O--, S--or N-thiazolinyl; SOCH ₃SO ₂CH ₃ONO ₂NO ₂N ₃NH ₂Heterocyclylalkyl; The heterocycle alkaryl; Aminoalkyl amino; Poly-alkyl amino; The silicyl that replaces; RNA cuts group; Reporter group; Intercalating agent; Be used to improve the group of the pharmacokinetic property of oligonucleotide; Or be used to improve the group of drug effect character of oligonucleotide and other substituents with similar quality.The preferred modification comprises 2'-methoxy ethoxy [2'-O-CH ₂CH ₂OCH ₃, be also referred to as the 2'-O-(2-methoxy ethyl)] (people such as Martin, (1995) Helv. Chim. Acta, 78,486).Other are preferably modified and comprise 2'-methoxyl group (2'-O--CH ₃), 2'-propoxyl group (2'-OCH ₂CH ₂CH ₃) and 2'-fluorine (2'-F).Similar modification can also be carried out on other positions on the oligonucleotide, particularly the 5' position of the 3' position of the sugar on the 3' terminal nucleotide and 5' terminal nucleotide.Oligonucleotide can also have sugared analogies for example cyclobutyl replace penta furyl glycosyl.

Oligonucleotide can also be in addition or is comprised that alternately nuclear base (being called " base " in the art usually for short) is modified or replacement.As used herein, " unmodified " or " natural " nucleotide comprises adenine (A), guanine (G), thymus pyrimidine (T), cytosine (C) and uracil (U).Modified nucleotide only comprises in natural acid rarely or the nucleotide of temporarily finding, hypoxanthine for example, 6-methyladenine, 5-Me pyrimidine particularly 5-methylcytosine (is also referred to as 5-methyl-2' deoxidation cytosine, and be commonly called 5-Me-C in the art), 5-hydroxymethyl cytosine (HMC), glycosyl HMC and gentiobiose base HMC, and synthesizing ribonucleotide, for example 2-aminoadenine, the 2-(methylamino) adenine, 2-(imidazole radicals alkyl) adenine, 2-(aminoalkyl amino) adenine or other assorted alkyl adenine that replaces, the 2-thiouracil, 2-sulfur thymus pyrimidine, 5-bromouracil, 5-hydroxylmethyluracil, guanozola, the 7-deazaguanine, N ₆(the amino hexyl of 6-) adenine and 2, the 6-diaminopurine.Kornberg, A., DNA Replication, W. H. Freeman ﹠ Co., San Francisco, 1980, the 75-77 pages or leaves; Gebeyehu, G. waits people (1987) Nucl. Acids Res., 15:4513).Can comprise for example inosine of " general " known in the art base.The 5-Me-C displacement has shown makes nucleic acid duplex stability increase 0.6-1.2 ℃.(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., editor, Antisense Research and Applications, CRC Press, Boca Raton, 1993, the 276-278 pages or leaves), and be at present preferred base substitution.

The another kind of oligonucleotide of the present invention is modified to relate to oligonucleotide is connected with one or more parts or conjugate chemistry, and described part or conjugate strengthen the activity or the cellular uptake of oligonucleotide.This type of part includes but not limited to lipid part, for example cholesterol moiety, cholesteryl part (people such as Letsinger, (1989) Proc. Natl. Acad. Sci. USA, 86,6553), cholic acid (people (1994) Bioorg. Med. Chem. Let. such as Manoharan, 4,1053), thioether hexyl-S-three beneze methane thiols (people (1992) Ann. N.Y. Acad. Sci. such as Manoharan, 660,306 for example; People such as Manoharan (1993) Bioorg. Med. Chem. Let., 3,2765), sulfydryl cholesterol (people such as Oberhauser, (1992) Nucl. Acids Res., 20,533), aliphatic chain for example dodecanediol or undecyl residue (people (1991) such as Saison-Behmoaras EMBO J. 1991,10,111; People such as Kabanov (1990) FEBS Lett., 259,327; People such as Svinarchuk (1993) Biochimie, 75,49), phospholipid two-cetyl-racemization-glycerol or 1 for example, 2-two-O-cetyl-racemization-glycerol-3-H-phosphine triethylenetetraminehexaacetic acid ammonium (people (1995) such as Manoharan Tetrahedron Lett., 36,3651; People such as Shea (1990) Nucl. Acids Res., 18,3777), polyamine or polyglycol chain (people (1995) such as Manoharan Nucleosides ﹠amp; Nucleotides, 14,969) or adamantane acetic acid (people (1995) such as Manoharan Tetrahedron Lett., 36,3651).The method that the oligonucleotide and being used to that comprises lipophilic portion prepares this class oligonucleotide is known in the art, and for example U.S. Patent number 5,138,045,5,218,105 and 5,459,255.

All positions in the given oligonucleotide are not necessarily as one man modified, and in fact the above-mentioned modification above can be mixed in the single oligonucleotide, and even the single nucleoside in oligonucleotide on.The present invention has comprised that also it is the oligonucleotide of the chimeric oligonucleotide of definition as mentioned.

In another embodiment, nucleic acid molecules of the present invention is puted together with another kind of part, and described another kind of part includes but not limited to the acid of dealkalize yl nucleosides, polyethers, polyamine, polyamide, peptide, carbohydrate, lipid or poly hydrocarbon compound.Those skilled in the art will recognize that these molecules can be on the several position on sugar, base or the phosphate with any nucleotide that constitutes nucleic acid molecules in one or more the connection.

The oligonucleotide that uses according to the present invention can be easily and more solito be prepared by well-known solid phase synthesis technique.Be used for this type of synthetic equipment and comprise that by several manufacturers Applied Biosystems sells.Can also adopt and be used for this type of synthetic any other method; The reality of oligonucleotide is synthetic fully in those of ordinary skills' ability.The also well-known similar technique that is to use to be to prepare other oligonucleotide, for example thiophosphate and alkyl derivative.The inferior amide (amidites) and controlled pore glass (CPG) product of the also well-known modification that is to use similar technique and is obtained commercially, inferior amide (amidites) and/or CPG(that biological example element, fluorescein, acridine or psoralen are modified can be from Glen Research, Sterling VA obtains), with the oligonucleotide of synthesizing fluorescently labeled, biotinylated or other modifications, the oligonucleotide modified of cholesterol for example.

According to the present invention, use to modify and for example to use the LNA monomer with the effectiveness, specificity and the acting duration that strengthen oligonucleotide and widen route of administration and comprise present chemistry, (Uhlman such as MOE, ANA, FANA, PS for example, Deng people (2000) Current Opinions in Drug Discovery ﹠ Development, the 3rd volume No 2).This can reach by some monomers of replacing in the present oligonucleotide via the LNA monomer.The oligonucleotide that LNA modifies can have the size that is similar to parent compound or can be bigger or preferably littler.The oligonucleotide that preferred this type of LNA modifies comprise less than about 70%, be more preferably less than about 60%, most preferably less than about 50% LNA monomer, and its size is 25 nucleotide of about 5 –, 20 nucleotide of 12 – more preferably from about.

The preferred oligonucleotide main chain of modifying includes but not limited to thiophosphate, the chirality thiophosphate, phosphorodithioate, phosphotriester, the aminoalkyl phosphotriester, methyl and other phosphonate esters comprise 3' alkylene phosphonic acids ester and chiral phosphonate, phosphinate, phosphoramidate comprises amino phosphoramidate of 3'-and aminoalkyl phosphoramidate, the thion phosphoramidate, the thion alkyl phosphate, thion alkyl phosphotriester and boron phosphate ester (boranophosphates) with normal 3'-5' bonding, these 2'-5' connects analog, with have the reversing polar those, wherein the phase adjacency pair 3'-5' of nucleoside unit is connected with 5'-2' with 5'-3' or 2'-5'.Also comprise various salt, salt-mixture and free acid form.

Instruct the above-mentioned representative United States Patent (USP) that contains the phosphorus linkage preparation to include but not limited to U.S. Patent number 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; With 5,625,050, it is incorporated herein by reference separately.

The preferred modified oligonucleotide main chain that does not wherein comprise phosphorus atoms has such main chain, its by bonding between short-chain alkyl or cycloalkyl nucleoside, mix between hetero atom and alkyl or cycloalkyl nucleoside that bonding forms between bonding or one or more short chain hetero atoms or heterocycle nucleoside.These comprise those (part is formed by the sugar moieties of nucleoside) with morpholino bonding; Siloxane main chain; Sulfide, sulfoxide and sulfone main chain; Formacetyl and sulfo-formacetyl main chain; Methylene formacetyl and sulfo-formacetyl main chain; Contain the chain hydrocarbon main chain; Sulfamate backbone; Methylene imino group and methylene diazanyl main chain; Sulphonic acid ester and sulfonamide backbone; Amide backbone; And have blended N, O, S and CH ₂Other of component part.

Instruct the representative United States Patent (USP) of above-mentioned oligonucleoside preparation to include but not limited to U.S. Patent number 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; With 5,677,439, it is incorporated herein by reference separately.

In other preferred oligonucleotide mimetics, bonding is that main chain is replaced by novel group between the sugar of nucleotide unit and nucleoside.Keeping these base units is used for and the hybridization of suitable nucleic acid target compound.A kind of this type of oligomeric compounds has shown the oligonucleotide mimetic with splendid hybridization character, is called as peptide nucleic acid(PNA) (PNA).In the PNA chemical compound, the sugar backbone of oligonucleotide is replaced by the amide containing main chain, particularly the aminoethyl glycine main chain.The nuclear base be retained and with direct or indirect combination of aza nitrogen atom of the amide moieties of main chain.The representative United States Patent (USP) of instruction PNA compound includes but not limited to U.S. Patent number 5,539,082; 5,714,331; With 5,719,262, it is incorporated herein by reference separately.The further instruction of PNA chemical compound can be people such as Nielsen, (1991) Science, 254, find among the 1497-1500.

In another preferred embodiment of the present invention, have the thiophosphate main chain oligonucleotide and have the oligonucleoside of hetero atom main chain, and particularly be called methylene (methyl-imino) or MMI main chain-CH ₂-NH-O-CH ₂-,-CH ₂-N(CH ₃)-O-CH ₂-,-CH ₂-O-N(CH ₃)-CH ₂-,-CH ₂N(CH ₃)-N(CH ₃) CH ₂-and-O-N(CH ₃)-CH ₂-CH ₂-, wherein the natural phosphodiester main chain is expressed as the O-P-O-CH of U.S. Patent number 5,489,677 mentioned above ₂-and the amide backbone of U.S. Patent number mentioned above 5,602,240.The oligonucleotide that further preferably has the morpholino backbone structure of U.S. Patent number 5,034,506 mentioned above.

Modified oligonucleotide can also comprise one or more replacement sugar moieties.Preferred oligonucleotide comprises one of the following: OH on the 2' position; F; O-, S-or N-alkyl; O-, S-or N-thiazolinyl; O-, S-or N-alkynyl; Or O alkyl-O-alkyl, wherein alkyl, thiazolinyl and alkynyl can be to replace or unsubstituted C to CO alkyl or C ₂To CO thiazolinyl and alkynyl.Particularly preferably be O(CH ₂) _nO _mCH ₃, O(CH ₂) _n, OCH ₃, O(CH ₂) _nNH ₂, O(CH ₂) nCH ₃, O(CH ₂) _nONH ₂And O(CH _2nON(CH ₂) _nCH ₃) ₂, wherein n and m can be 1 – about 10.Other preferred oligonucleotide comprise one of the following: C to CO, (low alkyl group of low alkyl group, replacement, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH on the 2' position ₃, OCN, Cl, Br, CN, CF ₃, OCF ₃, SOCH ₃, SO ₂CH ₃, ONO ₂, NO ₂, N ₃, NH ₂, silicyl, RNA cutting group, reporter group, the intercalating agent of Heterocyclylalkyl, heterocycle alkaryl, aminoalkyl amino, poly-alkyl amino, replacement, the group or be used to that is used to improve the pharmacokinetic property of oligonucleotide improve the group of drug effect character of oligonucleotide and other substituents with similar quality.The preferred modification comprises 2'-methoxy ethoxy (2'-O-CH ₂CH ₂OCH ₃, be also referred to as the 2'-O-(2-methoxy ethyl) or 2'-MOE) (people such as Martin, (1995) Helv. Chim. Acta, 78,486-504), i.e. alkoxyl alkoxy base.Further preferred the modification comprises 2'-dimethylamino oxygen base oxethyl, i.e. O(CH ₂) ₂ON(CH ₃) ₂Group is also referred to as 2'-DMAOE, as this paper hereinafter described in the embodiment and 2'-dimethylamino ethoxy ethyoxyl (this area is also referred to as 2'-O-dimethylamino ethoxy ethyl or 2'-DMAEOE), i.e. 2'-O-CH ₂-O-CH ₂-N(CH ₂) ₂

Other are preferably modified and comprise 2'-methoxyl group (2'-O CH ₃), amino propoxyl group (the 2'-O CH of 2'- ₂CH ₂CH ₂NH ₂) and 2'-fluorine (2'-F).Similar modification can also be carried out on other positions on the oligonucleotide, particularly in the 3' position of sugar on the 3' terminal nucleotide or connect in the oligonucleotide and the 5' position of 5' terminal nucleotide at 2'-5'.Oligonucleotide can also have sugared analogies for example cyclobutyl moiety replace penta furyl glycosyl sugar.Instruct this type of representative United States Patent (USP) of modifying sugared works preparation to include but not limited to U.S. Patent number 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; With 5,700,920, it is incorporated herein by reference separately.

Oligonucleotide can also comprise that nuclear base (being called " base " in the art usually for short) is modified or replacement.As used herein, " unmodified " or " natural " nucleotide comprises purine base adenine (A) and guanine (G), and pyrimidine bases thymus pyrimidine (T), cytosine (C) and uracil (U).Modified nucleotide comprises other synthetic and natural nucleotide, for example 5-methylcytosines (5-Me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, the 2-aminoadenine, the 6-methyl of adenine and guanine and other alkyl derivatives, the 2-propyl group of adenine and guanine and other alkyl derivatives, the 2-thiouracil, 2-sulfur thymus pyrimidine and 2-sulfur cytosine, 5-halogen uracil and cytosine, 5-propinyl uracil and cytosine, 6-azo uracil, cytosine and thymus pyrimidine, 5-uracil (pseudouracil), the 4-thiouracil, the 8-halogen, 8-amino, 8-mercaptan, 8-sulfane base, adenine and guanine that 8-hydroxyl and other 8-replace, the 5-halogen is the 5-bromine particularly, uracil and cytosine that 5-trifluoromethyl and other 5-replace, 7-methyl guanine (methylquanine) and 7-methyladenine, guanozola and 8-azaadenine, 7-deazaguanine and 7-denitrogenation adenine, and 3-deazaguanine and 3-denitrogenation adenine.

Further, nucleotide comprises and is disclosed in U.S. Patent number 3,687, in 808 those, be disclosed in ' The Concise Encyclopedia of Polymer Science And Engineering', the 858-859 page or leaf, Kroschwitz, J.I., ed. John Wiley and Sons, in 1990 those, by people such as Englisch, ' Angewandle Chemie, International Edition', 1991,30, the 613rd page of those disclosed and by Sanghvi, Y.S., the 15th chapter, ' Antisense Research and Applications', 289-302 page or leaf, Crooke, S.T. and Lebleu, B. ea., CRC Press, 1993 those disclosed.In these nucleotide some are used in particular for increasing the binding affinity of oligomeric compounds of the present invention.These comprise pyrimidine, 6-aza-pyrimidine and N-2, the N-6 of 5-replacement and the purine that 0-6 replaces, and comprise 2-aminopropyl adenine, 5-propinyl uracil and 5-propinyl cytosine.The 5-methylcytosine replacement has shown makes nucleic acid duplex stability increase 0.6-1.2 ℃ of (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., editor, ' Antisense Research and Applications', CRC Press, Boca Raton, 1993, the 276-278 pages or leaves), and be at present preferred base substitution, special more when the time with the sugar-modified combination of 2'-O-methoxy ethyl.

Instruct the representative United States Patent (USP) of above-mentioned modified nucleotide and other modified nucleotides preparation to include but not limited to U.S. Patent number 3,687,808 and 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692 and 5,681,941, it is incorporated herein by reference separately.

The another kind of oligonucleotide of the present invention is modified to relate to oligonucleotide is connected with one or more parts or conjugate chemistry, and described part or conjugate strengthen activity, cell distribution or the cellular uptake of oligonucleotide.

This type of part includes but not limited to for example cholesterol moiety (people such as Letsinger, (1989) of lipid part Proc. Natl. Acad. Sci. USA, 86,6553-6556), cholic acid (people such as Manoharan, (1994) Bioorg.Med. Chem. Let., 4,1053-1060), thioether hexyl-S-three beneze methane thiols (people such as Manoharan, (1992) Ann. N. Y. Acad. Sci., 660,306-309 for example; People such as Manoharan, (1993) Bioorg. Med. Chem. Let., 3,2765-2770), sulfydryl cholesterol (people such as Oberhauser, (1992) Nucl. Acids Res., 20,533-538), aliphatic chain for example dodecanediol or undecyl residue (people such as Kabanov, (1990) FEBS Lett., 259,327-330; People such as Svinarchuk, (1993) Biochimie, 75,49-54), phospholipid two-cetyl-racemization-glycerol or 1 for example, 2-two-O-cetyl-racemization-glycerol-3-H-phosphine triethylenetetraminehexaacetic acid ammonium (people such as Manoharan, (1995) Tetrahedron Lett., 36,3651-3654; People such as Shea, (1990) Nucl. Acids Res., 18,3777-3783), polyamine or polyglycol chain (people (1995) Nucleosides ﹠amp such as Manoharan; Nucleotides, 14,969-973) or adamantane acetic acid (people such as Manoharan, (1995) Tetrahedron Lett., 36,3651-3654), palmityl part (people such as Mishra, (1995) Biochim. Biophys. Acta, 1264,229-237) or 18-amine. or hexyl amino-carbonyl-t oxycholesterol part (people such as Crooke, (1996) J. Pharmacol. Exp. Ther., 277,923-937).

Instruct the representative United States Patent (USP) of this class oligonucleotide conjugate preparation to include but not limited to U.S. Patent number 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241,5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, it is incorporated herein by reference separately.

Drug development: chemical compound of the present invention can also be applied to drug development and target is confirmed the field.The present invention is included in and uses this paper compounds identified and preferred target area section in the drug development effort, to be illustrated in the relation that exists between lipid transfer and metabolic gene polynucleotide and morbid state, phenotype or the condition of illness.These methods comprise detection or regulate lipid transfer and metabolic gene polynucleotide, it comprises makes sample, tissue, cell or biology contact with chemical compound of the present invention, some time measures nucleic acid or protein level and/or the relevant phenotype or the chemical end of the final point of lipid transfer and metabolic gene polynucleotide and randomly makes measured value with non-processing sample or with the sample comparison of further compound treatment of the present invention after treatment.These methods can also be tested parallel with other or combination is carried out, and are used for target with the function of measuring unknown gene and confirm process, or measure the specific gene product as the effectiveness that is used for the treatment of or prevents the target of specified disease, condition of illness or phenotype.

The rise or the inhibition of assessment gene expression:

Exogenous nucleic acid is transferred in host cell or the biology and can be assessed by the existence of direct detection cell or biological amplifying nucleic acid.This type of detection can reach by several method well-known in the art.For example, the existence of exogenous nucleic acid can use the primer of the specific amplification nucleotide sequence relevant with nucleic acid to detect by southern blotting technique or by polymerase chain reaction (PCR) technology.The expression of exogenous nucleic acid can also use conventional method to measure, and comprises gene expression analysis.For example, the mRNA that is produced by exogenous nucleic acid can use RNA trace and reverse transcription PCR (RT-PCR) to detect and quantitatively.

Rna expression from exogenous nucleic acid also can be by measuring enzymatic activity or reporting that protein active detects.For example, antisense is regulated active minimizing or the increase that can be used as in the target nucleic acid expression and is measured indirectly, produces the indication of effector RNA as exogenous nucleic acid.Based on sequence conservation, can design primer and be used for the coding region of amplified target gene.At first, the coding region of expressing from every kind of gene topnotch can be used to make up the model crt gene, although can use any coding or noncoding region.Every kind of crt gene assembles by each coding region is inserted between report coding region and the polyadenylic acid signal thereof.These plasmids have the reporter gene in upstream region of gene part and the mRNA of the potential rna i target in the 3' noncoding region with generation.The effectiveness of indivedual antisense oligonucleotides will be assessed by the adjusting of reporter gene.Useful in the method for the invention reporter gene comprises acetohydroxy acid synthase (AHAS), alkali phosphatase (AP), beta galactosidase (LacZ), β glycuronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescence protein (YFP), blue fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS) and derivant thereof.Multiple selectable marker is obtainable, and it gives the resistance for ampicillin, bleomycin, chloromycetin, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphine oxamate, puromycin and tetracycline.The method of measuring the reporter gene adjusting is well-known in the art, and includes but not limited to fluorescence method (for example fluorescent spectrometry, fluorescence-activated cell sorting (FACS), fluorescence microscopy), antibiotic resistance mensuration.

Lipid transfer and metabolic gene protein and mRNA express and can use the methods analyst that those skilled in the art are known and other place of this paper is described.For example, immunoassay such as ELISA can be used for measuring protein level.Be used for the lipid transfer of ELISAs and metabolic gene antibody can from for example R﹠D Systems (Minneapolis, MN), Abcam, Cambridge, MA buys.

In each embodiment, lipid transfer in the sample of handling with antisense oligonucleotide of the present invention (for example in the body or cell in vitro or tissue) and metabolic gene express (for example mRNA or protein) by with control sample in lipid transfer and metabolic gene expression ratio estimate.For example, protein or expression of nucleic acids can compare with method known to those skilled in the art and simulation process or untreated samples.Alternatively, carry out with can depend on information needed with the comparison of the sample that contrasts the antisense oligonucleotide processing (for example, having change or different sequence).In another embodiment, processing can compare with handling the difference of sample in expressing with respect to different nucleic acid in the untreated samples (comprising any standard that researcher sees fit, for example house-keeping gene) with respect to the difference in lipid transfer in the untreated samples and metabolic gene protein or the expression of nucleic acid.

The difference of observing can represent by hope, for example, with than or fractional form, be used for and compare.In each embodiment, with respect to untreated samples or the sample handled with contrast nucleic acid, lipid transfer and metabolic gene mRNA or proteinic level increase or reduce about 1.25-doubly to about 10-times or more in the sample of handling with antisense oligonucleotide of the present invention.In each embodiment, lipid transfer and metabolic gene mRNA or proteinic level increase or reduce at least about 1.25-doubly, at least about 1.3-doubly, at least about 1.4-doubly, at least about 1.5-doubly, at least about 1.6-doubly, at least about 1.7-doubly, at least about 1.8-doubly, at least about 2-doubly, at least about 2.5-doubly, at least about 3-doubly, at least about 3.5-doubly, at least about 4-doubly, at least about 4.5-doubly, at least about 5-doubly, at least about 5.5-doubly, at least about 6-doubly, at least about 6.5-doubly, at least about 7-doubly, at least about 7.5-doubly, at least about 8-doubly, at least about 8.5-doubly, at least about 9-doubly, at least about 9.5-doubly, at least about 10-times or more.

Test kit, research reagent, diagnosis and treatment

Chemical compound of the present invention can be used for diagnosis, treatment and prevention, and as research reagent and reagent constituents.In addition, can be used to illustrate the function of specific gene usually by those of ordinary skills, or distinguish the various members' of biological approach function with the antisense oligonucleotide of strong specific inhibition of gene expression.

For the use in test kit and diagnosis and in various biosystems, chemical compound of the present invention makes up as the instrument in differentiation and/or the combinative analysis separately or with other chemical compounds or therapeutic agent, to be illustrated in the expression pattern of the cell and the partial or complete complement of the interior gene of expressing of tissue.

As used herein, term " biosystem " or " system " are defined as any biology, cell, cell culture or tissue, its expression or make it possible to express the product of lipid transfer and metabolic gene.These include but not limited to people, transgenic animal, cell, cell culture, tissue, xenograft, graft and combination thereof.

As a non-limitative example, expression pattern in the cell or tissue of handling with one or more antisense compounds compares with control cells or the tissue do not handled with antisense compounds, and the pattern that produces is analyzed with regard to the level of difference of gene expression, because they for example relate to disease association, signal pathway, celluar localization, expression, size, structure or the function that is examined gene.These analyses can be to stimulating or irritation cell and in the existence of other chemical compounds that influences expression pattern or execution not.

The example of the method for gene expression analysis known in the art comprises DNA array or microarray (Brazma and Vilo, (2000) FEBS Lett., 480,17-24; Celis waits the people, (2000) FEBS Lett., 480,2-16), (Madden waits the people, (2000) for the serial analysis of SAGE(gene expression Drug Discov. Today, 5,415-425), the amplification of the Restriction Enzyme of READS(digested cdna s) (Prashar and Weissman, (1999) Methods Enzymol., 303,258-72), the total gene expression analysis of TOGA() (Sutcliffe waits the people, (2000) Proc. Natl. Acad. Sci. U.S.A., 97,1976-81), (Celis waits the people, (2000) for protein array and proteomics FEBS Lett., 480,2-16; Jungblut waits the people, (1999) Electrophoresis, 20, expressed sequence labelling (EST) checks order 2100-10),, and (Celis waits the people, (2000) FEBS Lett., 480,2-16; Larsson waits the people, (2000) J. Biotechnol., 80,143-57), (Fuchs waits the people, (2000) to subdue RNA fingerprinting (SuRF) Anal. Biochem., 286,91-98; Larson waits the people, (2000) Cytometry, 41,203-208), subdue clone, difference and show (DD) (Jurecic and Belmont, (2000) Curr. Opin. Microbiol., 3,316-21), (Carulli waits the people to comparative genome hybridization, (1998) J. Cell Biochem. Suppl., 31,286-96), the FISH(fluorescence in situ hybridization) technology (Going and Gusterson, (1999) Eur. J. Cancer, 35,1895-904) and mass spectrography (To, Comb.(2000) Chem. High Throughput Screen, 3,235-41).

Chemical compound of the present invention for research and diagnose useful because the nucleic acid hybridization of these chemical compounds and coding lipid transfer and metabolic gene.For example, with this type of efficient and as hybridizing to become the oligonucleotide of effective lipid transfer and metabolic gene regulator under this type of condition disclosed herein, respectively effective primer or probe under the condition that helps gene amplification or detection.These primers and probe are used for the method for specific detection of nucleic acid molecules of requirement coding lipid transfer and metabolic gene and the amplification that is used for described nucleic acid molecules and are used to detect or are used for using in the further research of lipid transfer and metabolic gene.The antisense oligonucleotide of the present invention particularly hybridization of the nucleic acid of primer and probe and coding lipid transfer and metabolic gene can detect by methods known in the art.These class methods can comprise radioactive label or any other suitable detection method of the puting together of enzyme and oligonucleotide, oligonucleotide.Can also prepare and use this type of detection method to be used for the lipid transfer of test sample and the test kit of metabolic gene level.

The specificity and the sensitivity of antisense also are used for the treatment of purposes by those skilled in the art.Antisense compounds is used as the treatment part in animal comprises morbid state treatment among the people.The antisense oligonucleotide medicine is applied to the people safely and effectively, and numerous clinical trial is underway at present.Therefore determine that antisense compounds can be useful treatment pattern, it can be configured in that to be used for the treatment of cell, tissue and animal particularly useful in people's the therapeutic scheme.

For treatment, suspect that have can lipid transfer and metabolic gene polynucleotide be expressed the disease for the treatment of or the preferred people of animal of disease treats by using according to antisense compounds of the present invention by regulating.For example, in a non-limiting embodiments, this method comprises to the lipid transfer of the animal administering therapeutic effective dose of needs treatment and the step of metabolic gene regulator.The activity that lipid transfer of the present invention and metabolic gene regulator are effectively regulated lipid transfer and metabolic gene, or regulate lipid transfer and metabolic gene protein expression.In one embodiment, and compare lipid transfer and metabolic gene active or express and be suppressed about 10% in animal.Preferably, the active or expression of lipid transfer and metabolic gene is suppressed about 30% in animal.More preferably, the active or expression of lipid transfer and metabolic gene is suppressed 50% or more in animal.Therefore, with compare, oligomeric compounds makes the expression of lipid transfer and metabolic gene mRNA regulate at least 10%, at least 50%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100%.

In one embodiment, and compare lipid transfer and metabolic gene active or express and increase about 10% in animal.Preferably, the active or expression of lipid transfer and metabolic gene increases about 30% in animal.More preferably, the active or expression of lipid transfer and metabolic gene increases by 50% or more in animal.Therefore, with compare, oligomeric compounds makes the expression of lipid transfer and metabolic gene mRNA regulate at least 10%, at least 50%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100%.

For example, the expression decreased of lipid transfer and metabolic gene can be measured in animal serum, blood, fatty tissue, liver or any other body fluid, tissue or organ.Preferably, the cell that comprises in described liquid, tissue or the organ analyzed comprises nucleic acid molecules and/or the lipid transfer and the metabolic gene protein self of coding lipid transfer and metabolic gene peptide.

Add in the suitable pharmaceutically acceptable diluent or carrier by the chemical compound with effective dose, chemical compound of the present invention can be used for pharmaceutical composition.The use of Compounds and methods for of the present invention also can be that prevention is gone up useful.

Conjugate

The another kind of oligonucleotide of the present invention is modified to relate to oligonucleotide is connected with one or more parts or conjugate chemistry, and described part or conjugate strengthen activity, cell distribution or the cellular uptake of oligonucleotide.These parts or conjugate can comprise with functional group for example uncle or the covalently bound conjugate group of secondary hydroxyl group.Conjugate group of the present invention comprise intercalating agent, reporter molecule, polyamine, polyamide, Polyethylene Glycol, polyethers, enhancing oligomer drug effect character group and strengthen the group of the pharmacokinetic property of oligomer.General conjugate group comprises cholesterol, lipid, phospholipid, biotin, azophenlyene, folic acid, phenanthridines, anthraquinone, acridine, fluorescein, rhodamine, coumarin and dyestuff.In background of the present invention, the group that strengthens drug effect character comprises and improves picked-up, strengthens the group that the sequence-specific of the resistance of degraded and/or reinforcement and target nucleic acid is hybridized.In background of the present invention, the group that strengthens pharmacokinetic property comprises the group of the picked-up, distribution, metabolism or the discharge that improve chemical compound of the present invention.Representative conjugate group is disclosed among the international patent application no PCT/US92/09196 and U.S. Patent number 6,287,860 that submitted on October 23rd, 1992, and it is incorporated herein by reference.Conjugate partly includes but not limited to for example cholesterol moiety, cholic acid, thioether hexyl-S-three beneze methane thiols, sulfydryl cholesterol, aliphatic chain dodecanediol or undecyl residue, phospholipid two-cetyl-racemization-glycerol or 1 for example for example for example of lipid part, 2-two-O-cetyl-racemization-glycerol-3-H-phosphine triethylenetetraminehexaacetic acid ammonium, polyamine or polyglycol chain or adamantane acetic acid, palmityl part or 18-amine. or hexyl amino-carbonyl-oxycholesterol part.Oligonucleotide of the present invention can also be puted together with the active medicine material, for example aspirin, warfarin, Phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, red sarcosine, 2,3,5-Triiodobenzoic acid, flufenamic acid, folinic acid, benzothiadiazine, chlorothiazide, diaza

, indomethacin (indomethicin), barbiturate, cephalosporin, sulphonamides, antidiabetic drug, antibacterial or antibiotic.

Instruct the representative United States Patent (USP) of this class oligonucleotide conjugate to include but not limited to U.S. Patent number 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241,5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

Preparation

Chemical compound of the present invention can also mix, encapsulate, put together or otherwise combine with the mixture of other molecules, molecular structure or chemical compound, as for example liposome, the receptor target molecule, per os, rectum, part or other preparations are used for helping picked-up, distribute and/or absorb.The representative United States Patent (USP) of instructing this type of picked-up, distribute and/or absorb the auxiliary agent preparation includes but not limited to U.S. Patent number 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,165; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; With 5,595,756, it is incorporated herein by reference separately.

Although antisense oligonucleotide need not to use in the background of carrier, so that regulating target expresses and/or function, but embodiment of the present invention relate to the expression vector establishment body that is used for the antisence oligonucleotide, comprise promoter, hybrid promoter gene order, and have strong constitutive promoter activity, or can be under required situation inductive promoter activity.

In one embodiment, the present invention practice relates at least a with in the suitable aforementioned antisense oligonucleotide of delivery of nucleic acids systemic application.In one embodiment, this system comprises the non-virus carrier that is operably connected with polynucleotide.The example of this type of non-virus carrier comprises separately (for example, any one among the SEQ ID NO:23 – 263 or a plurality of) or the oligonucleotide that makes up with suitable protein, polysaccharide or lipid formulations.

Suitable in addition delivery of nucleic acids system comprises viral vector, generally is from least a sequence in following: the haemagglutinating virus of adenovirus, adeno-associated virus (AAV), helper virus dependent form adenovirus, retrovirus or Japanese liposome (HVJ) complex.Preferably, viral vector comprises the strong eukaryotic promoter that is operably connected with polynucleotide, for example cytomegalovirus (CMV) promoter.

Preferred in addition carrier comprises viral vector, fusion rotein and chemically conjugated thing.Retroviral vector comprises Moloney muroid leucovirus and based on the virus of HIV.A kind ofly preferably comprise at least 2 kinds of carriers based on the viral vector of HIV, wherein gag and pol gene be from the HIV genome, and EnvGene is from another kind of virus.Dna viral vector is preferred.These carriers comprise for example for example herpes simplex I virus (HSV) carrier [Geller, people such as A.I., (1995) of vaccinia subgroup virus or Fowlpox virus vector, herpesvirus vector of poxvirus vector J. Neurochem, 64:487; Lim, F. waits the people, DNA Cloning:Mammalian Systems, D. Glover, Ed.(Oxford Univ. Press, Oxford England) (1995); Geller, people such as A.I., (1993) Proc Natl. Acad. Sci.:U.S.A.:90 7603; Geller, A.I. waits the people, (1990) Proc Natl. Acad. SciUSA:87:1149], adenovirus vector (people such as LeGal LaSalle, Science, 259:988(1993); Davidson waits the people, (1993) Nat. Genet.3:219; Yang waits the people, (1995) J. Virol. 69:2004) and gland relevant viral vector (Kaplitt, M.G. wait the people, (1994) Nat. Genet. 8:148).

Antisense compounds of the present invention comprises salt or any other chemical compound of any pharmaceutically acceptable salt, ester or this type of ester, and it can provide (directly or indirectly) biologic activity metabolite or its remnants being applied to after animal comprises the people.

Term " pharmaceutically acceptable salt " refers to the physiology and the pharmaceutically acceptable salt of The compounds of this invention: promptly keep the required biological activity of parent compound and it is not given the salt of undesirable toxicological effect.For oligonucleotide, preferred example of pharmaceutically acceptable salt and uses thereof is at U.S. Patent number 6,287, further describes in 860, and it is incorporated herein by reference.

The present invention also comprises pharmaceutical composition and the preparation that comprises antisense compounds of the present invention.Pharmaceutical composition of the present invention can be used in many ways, and this depends on needs part or systemic treatment and depend on zone to be treated.Use can be partial (comprise eye and comprise that to mucosa vagina and rectum send), lung for example by powder or aerocolloidal suction or be blown into, comprise and pass through aerosol apparatus; In the trachea, intranasal, epidermis and percutaneous), per os or parenteral.Parenteral administration comprises intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; Or intracranial, for example use in the sheath or in the ventricle.

In order to handle the tissue among the central nervous system, administration can be undertaken by for example injecting or be infused in the cerebrospinal fluid.The administration of antisense RNA in cerebrospinal fluid described in U.S. Patent Application Publication No. 2007/0117772 for example, the method for familial ALS progression of disease " be used for slow down ", and its integral body is incorporated this paper into as a reference.

When intention antisense oligonucleotide of the present invention gave cell among the central nervous system, administration can be used and can be promoted theme antisense oligonucleotide infiltration one or more reagent by blood brain barrier to carry out.Injection can for example be carried out in the Hippocampus entorhinal cortex.Neurotrophic factor is described in for example U.S. Patent number 6,632,427, " the adenovirus vector-mediated gene transfer to the oblongata motor neuron " by using adenovirus vector sending of motor neuron in the muscular tissue, and it incorporates this paper into as a reference.Carrier to brain for example directly sending in striatum, thalamus, Hippocampus or the black substance be known in the art, and be described in for example U.S. Patent number 6,756,523, " be used for transporting the adenovirus vector of exogenous gene to central nervous system's especially brain cell ", it incorporates this paper into as a reference.Using can be fast, as by injection, or carries out in one period, as by slow infusion or use slow delivery formulations.

The theme antisense oligonucleotide can be connected or coupling with the reagent that expectation pharmacy or pharmacokinetics character are provided.For example, antisense oligonucleotide can be striden any material coupling of blood brain barrier infiltration or transhipment with promotion known in the art, such as the antibody of TfR, and uses by intravenous injection.Antisense compounds can be connected with viral vector, and for example, it makes that antisense compounds is more effective and/or increases the transhipment that antisense compounds is striden blood brain barrier.The infiltration blood-brain barrier disruption also can be realized like this; by for example infusion sugar; include but not limited to the meso erithritol; xylitol; D (+) galactose; D (+) lactose; D (+) xylose; galactitol; inositol; L (-) fructose; D (-) mannitol; D (+) glucose; D (+) arabinose; D (-) arabinose; cellobiose; D (+) maltose; D (+) Raffinose; L (+) rhamnose; D (+) 6-(.alpha.-D-galactosido)-D-glucose.; D (-) ribose; ribitol; D (+) 1,2,3,4,5-pentanepentol; L (-) 1,2,3,4,5-pentanepentol; D (+) fucose; L (-) fucose; D (-) lyxose; L (+) lyxose and L (-) lyxose; or aminoacid, include but not limited to glutamine; lysine; arginine; agedoite; aspartic acid; cysteine; glutamic acid; glycine; histidine; leucine; methionine; phenylalanine; proline; serine; threonine; tyrosine; valine and taurine.The method and the material that are used to increase the blood brain barrier infiltration are described in for example U.S. Patent number 4,866,042 " being used to carry genetic stocks to stride the method for blood brain barrier ", 6,294,520 " being used for material " and 6 by blood brain barrier, in 936,589 " the parenteral induction systems ", they all integral body incorporate this paper into as a reference.

The theme antisense compounds can for example liposome, receptor target molecule, oral, rectum, part or other preparations mix, seal, coupling or be connected in addition with the mixture of other molecules, molecular structure or chemical compound, are used for promoting picked-up, distribute and/or absorb.For example, cation lipid can be included in the preparation to promote the oligonucleotide picked-up.A kind of such compositions that show to promote picked-up be LIPOFECTIN (can derive from GIBCO-BRL, Bethesda, MD).

Oligonucleotide with at least one 2'-O-methoxy ethyl modification is considered to particularly useful for dosage forms for oral administration.The pharmaceutical composition and the preparation that are used for local application can comprise percutaneous patch, ointment, lotion, emulsifiable paste, gel, drop, suppository, spray, liquid and powder.Conventional pharmaceutical carrier, water, powder or oil base, thickening agent etc. can be to need or hope.Bag also can be useful by condom, glove etc.

The pharmaceutical preparation of the present invention that can present with unit dosage forms easily can be prepared according to well-known routine techniques in the pharmaceutical industries.This type of technology comprises makes active component and one or more pharmaceutical carriers or excipient reach bonded step.Generally speaking, preparation is prepared by following: active component and liquid-carrier or the meticulous solid carrier that separates or both are reached equably and closely combine, and product is shaped.

Compositions of the present invention can be mixed with many any in may dosage forms, such as but not limited to tablet, capsule, gel capsule, liquid syrups, soft gel, suppository and enema.Compositions of the present invention can also be formulated as suppository in water, non-water or blending agent.Water slurry can further comprise the material that increases suspension viscosity, comprises for example sodium carboxymethyl cellulose, Sorbitol and/or glucosan.Suspension can also comprise stabilizing agent.

Pharmaceutical composition of the present invention includes but not limited to solution, emulsion, foam and pharmaceutical preparations containing liposomes.Pharmaceutical composition of the present invention and preparation can comprise one or more penetration enhancers, carrier, excipient or other activity or non-active ingredient.

The emulsion droplet form that generally to be a kind of liquid surpass 0.1 μ m usually with diameter is dispersive heterogeneous body system in another kind.Emulsion removes decentralized photo can comprise other component, and the active drug that can be used as the solution in water, oil phase or himself exist mutually as separation.Microemulsion comprises as one embodiment of the invention.Emulsion and uses thereof is well-known in the art, and at U.S. Patent number 6,287, further describes in 860.

Preparation of the present invention comprises Liposomal formulation.As using in the present invention, term " liposome " means the vesicle of being made up of the amphiphilic lipids of arranging in one or more spherical bilayers.Liposome is the single or multiple lift vesicle, and it has the film that is formed by the lipophilic material and comprises the aqueous interior for the treatment of delivering compositions.Cationic-liposome is the liposome of positively charged, and it is considered to interact with electronegative dna molecular, to form stable compound.It is that pH sensitivity or electronegative liposome are considered to entrap DNA rather than compound with it.Cation and non-cationic liposome have been used for DNA is delivered to cell.

Liposome also comprises " stereoscopic stable " liposome, and as used herein, it is meant the term of the liposome that comprises one or more special lipids.In the time of in mixing liposome, these special lipids cause with respect to the liposome that lacks this type of special lipid, the liposome with enhanced cycle life.The example of the liposome of stereoscopic stable is that the part that the vesicle of wherein liposome forms lipid part comprises one or more glycolipids, or by one or more hydrophilic polymers for example Polyethylene Glycol (PEG) part deutero-those.Liposome and uses thereof is at U.S. Patent number 6,287, further describes in 860.

Pharmaceutical preparation of the present invention and compositions can also comprise surfactant.The purposes of surfactant in drug products, preparation and emulsion is well-known in the art.Surfactant and uses thereof is at U.S. Patent number 6,287, further describes in 860, and it is incorporated herein by reference.

In one embodiment, the present invention adopts various penetration enhancers, to realize particularly effectively sending of oligonucleotide of nucleic acid.Except that helping non-lipophilic drug diffusion to cross the cell membrane, penetration enhancers also strengthens the permeability of lipophilic medicine.Penetration enhancers can be categorized as and belong to one of 5 extensive categories, i.e. surfactant, fatty acid, bile salts, chelating agen and non-chelating non-surface-active agent.Penetration enhancers and uses thereof is at U.S. Patent number 6,287, further describes in 860, and it is incorporated herein by reference.

Those skilled in the art will recognize that more solito is that route of administration designs preparation according to its desired use.

The preferred formulation that is used for local application comprises those of oligonucleotide wherein of the present invention and local delivery reagent mix, and described local delivery reagent is lipid, liposome, fatty acid, fatty acid ester, steroid, chelating agen and surfactant for example.Preferred lipid and liposome comprise neutrality (for example two oleoyls-phosphatidyl DOPE ethanolamine, dimyristoyl phosphatidyl choline DMPC, distearoyl phosphatidylcholine), negative (for example GLYCEROL,DIMYRISTOYL PHOSPHATIDYL DMPG) and cationic (for example two oleoyl tetramethyl aminopropyl DOTAP and DOPE DOTMA).

Use for part or other, oligonucleotide of the present invention can be encapsulated in the liposome or can particularly form complex with cationic-liposome with it.Alternately, oligonucleotide can be particularly compound with cation lipid with lipid.Preferred fatty acid and ester, its pharmaceutically acceptable salt and uses thereof be at U.S. Patent number 6,287, further describes in 860.

Be used for the compositions of dosage forms for oral administration and preparation comprise powder or granule, microgranule, nano-particle, at suspension or solution, capsule, gel capsule, wafer, tablet or the small pieces of water or non-aqueous media.Thickening agent, flavoring agent, diluent, emulsifying agent, dispersing aid or bonding agent may be wished.Preferred per os preparation is that oligonucleotide wherein of the present invention combines those that use with one or more penetration enhancers, surfactant and chelating agen.Preferred surfactant comprises fatty acid and/or its ester or salt, bile acid and/or its salt.Preferred bile acid/salt and fatty acid and uses thereof be at U.S. Patent number 6,287, further describes in 860, and it is incorporated herein by reference.The further preferably combination of penetration enhancers is for example with the fatty acid/salt of bile acid/salt combination.Particularly preferred combination is lauric sodium salt, capric acid and UDCA.Further penetration enhancers comprises polyoxyethylene-9-Laurel ether, polyoxyethylene-20-cetyl ether.Oligonucleotide of the present invention can comprise the spray-dried granules oral delivery with particle form, or compound to form microgranule or nano-particle.Oligonucleotide chelating agent and uses thereof is at U.S. Patent number 6,287, further describes in 860, and it is incorporated herein by reference.

Be used in the parenteral, sheath or compositions and the preparation used in the ventricle can comprise aseptic aqueous solution, it can also comprise buffer agent, diluent and other appropriate addns, such as but not limited to penetration enhancers, carrier compound and other pharmaceutically acceptable carrier or excipient.

Particular of the present invention provides the pharmaceutical composition that comprises one or more oligomeric compounds and one or more other chemotherapeutants, and described other chemotherapeutants work by non-antisense mechanism.The example of this type of chemotherapeutant includes but not limited to cancer chemotherapeutic medicine, for example daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, darubicin, esorubicin, bleomycin, Mafosfamide, ifosfamide, cytosine arabinoside, two-chloroethyl-nitroso ureas, busulfan, ametycin, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, zitazonium, dacarbazine, procarbazine, altretamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, the methylcyclohexyl nitroso ureas, chlormethine, melphalan, cyclophosphamide, the 6-mercaptopurine, the 6-thioguanine, cytosine arabinoside, 5-azacytidine, hydroxyurea, pentostatin, 4-hydroxyl peroxide cyclophosphamide, 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, Irinotecan, hycamtin, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES).When using with chemical compound of the present invention, this type of chemotherapeutant is (for example 5-FU and oligonucleotide), (for example 5-FU and oligonucleotide a period of time in turn individually, be MTX and oligonucleotide subsequently) or make up (for example 5-FU, MTX and oligonucleotide, or 5-FU, X-ray therapy and oligonucleotide) with one or more other these type of chemotherapeutants and use.Anti-inflammatory agent includes but not limited to that NSAID (non-steroidal anti-inflammatory drug) and corticosteroid and antiviral agents include but not limited to ribavirin, vidarabine, acyclovir and ganciclovir, also can make up in compositions of the present invention.The combination of antisense compounds and other non-antisense drugs also within the scope of the invention.Two or more combination of compounds can use together or in turn.

In another related embodiment, one or more antisense compounds that compositions of the present invention can comprise first kind of nucleic acid of targeting are one or more other antisense compounds of oligonucleotide and second kind of nucleic acid target of targeting particularly.For example, first kind of target can be the specific antisense sequences of lipid transfer and metabolic gene, and second kind of target can be the zone from another kind of nucleotide sequence.Alternately, compositions of the present invention can comprise two or more antisense compounds of the zones of different of identical lipid transfer of targeting and metabolic gene nucleic acid target.Numerous examples of antisense compounds illustrate in this article, and other can be selected from suitable combination thing known in the art.Two or more combination of compounds can use together or in turn.

Administration:

The preparation of therapeutic combination and subsequent applications thereof (administration) are considered in art technology.Administration depends on the severity and the responsiveness of morbid state to be treated, uses the therapeutic process that continues a couple of days to the several months, or until the reduction that realizes curing or reaching morbid state.Best dosage regimen can be calculated by the intravital drug accumulation measurement of patient.Those of ordinary skill can easily determine optimal dose, medication and repetition rate.Optimal dose can depend on the relative potency of indivedual oligonucleotide and become, and generally can be based on finding in vitro and in vivo in the animal model effectively EC _50sEstimate.Generally speaking, dosage is 0.01 μ g-100 g/kg body weight, and can every day, weekly, every month or give one or many every year, or even per 2 – gave once in 20 years.Those of ordinary skills can easily estimate the repetition rate about administration, and this is based on the medicine time of staying and the concentration measured in body fluid or tissue.After successful treatment, can wish to make the patient experience maintenance therapy with prevent disease state recurrence, wherein oligonucleotide is used with maintenance dose, and scope is 0.01 μ g-100 g/kg body weight, once a day or repeatedly, by per 20 years once.

In each embodiment, the patient treats with following drug dose: at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 or at least about 100 mg/kg body weight.Some injected dose of antisense oligonucleotide is described in for example U.S. Patent number 7,563,884, " the antisense adjusting that PTP1B expresses ", and its integral body is incorporated this paper into as a reference.

Although above describing various embodiments of the present invention, be to be understood that they only as an example rather than the restriction present.Can carry out for openly numerous changes of embodiment according to this paper disclosure, and not deviate from the spirit or scope of the present invention.Therefore, range of the present invention and scope should not be subjected to any restriction in the above-mentioned embodiment.

The All Files that this paper mentions is incorporated herein by reference.All publications quoted among the application and patent document are incorporated herein by reference for all purposes, and its degree is pointed out identical so individually with each indivedual publication or patent document.By its quoting in the various lists of references of presents, the applicant does not admit that any concrete list of references is its invention " prior art ".The embodiment of the present composition and method illustrates in the following embodiments.

Embodiment

Following non-limiting example is used for illustrating selection embodiment of the present invention.Be to be understood that shown in ratio and the variation in the Res fungibiles in the component element will be conspicuous for those skilled in the art, and in the scope of embodiment of the present invention.

Embodiment 1: the design of antisense oligonucleotide, it is for having specificity with the nucleic acid molecules of lipid transfer and metabolic gene antisense and/or the sense strand of lipid transfer and metabolic gene polynucleotide

As mentioned above, the oligonucleotide that term " for ... special oligonucleotide " or " oligonucleotide target " refer to have following sequence: (i) can form stable compound, or (ii) can form stable duplex with the part of the mRNA transcript of target gene with the part of target gene.

The selection of suitable oligonucleotide obtains promoting that described computer program is compared nucleotide sequence automatically and pointed out homogeneity or the homology zone by the program of using a computer.The nucleotide sequence that this class method is used for relatively obtaining is for example by search database GenBank or by order-checking PCR product for example.The nucleotide sequence that relatively allows to select to show the suitable homogeneity degree between the species from the nucleotide sequence of a series of species.Under the situation of the gene that does not check order, carry out southern blotting technique to allow to measure the homogeneity degree between gene in target species and other species.By under various strict degree, carrying out southern blotting technique, as known in the art, can obtain the approximate measure of homogeneity.Such oligonucleotide allow is selected in these operations, its demonstrate with experimenter to be controlled in the height of target nucleic acid sequence complementary and with other species in corresponding nucleic sequence than the low degree complementarity.The technical staff will recognize that there is sizable move place in the appropriate area at the gene of selecting to be used for using in the present invention.

Antisense compounds is " but specific hybrid ", this moment, chemical compound was regulated to cause function and/or activity with the normal function that combining of target nucleic acid disturbed target nucleic acid, and the complementarity that has enough degree, needing therein to avoid the non-specific binding of antisense compounds and non-target nucleic acid sequence under the bonded condition of specificity, promptly under physiological condition and wherein, carrying out under the condition of measuring under the external test situation under mensuration or the therapeutic treatment situation in vivo.

The hybridization character of oligonucleotide described herein can be measured by one or more external tests as known in the art.For example, use melting curve to measure, can obtain the character of oligonucleotide described herein by the bond strength between mensuration target natural antisense and the potential drug molecule.

Any definite method of use measurement intermolecular interaction intensity for example melting curve is measured, and can estimate the bond strength between target natural antisense and the potential drug molecule (molecule).

Melting curve is measured the temperature of determining to take place for natural antisense/molecular complex down at it fast transition from the two strands to the single stranded conformational.This temperature is widely acknowledged to be the reliable measurements of 2 kinds of interaction strengths between the molecule.

Use the cDNA copy or synthetic DNA or the RNA nucleotide corresponding of actual natural antisense RNA molecule, can carry out melting curve and measure with the binding site of molecule.Comprising the plurality of reagents box that all that carry out this mensuration must reagent is obtainable (for example Applied Biosystems Inc. MeltDoctor test kit).These test kits comprise the suitable buffer solution that comprises one of double-stranded DNA (dsDNA) combination dye (for example ABI HRM dyestuff, SYBR Green, SYTO etc.).The character of dsDNA dyestuff is such, makes them send fluorescence hardly with free form, but is height fluorescence when combining with dsDNA.

In order to carry out mensuration, cDNA or corresponding oligonucleotide are mixed with the concentration that the scheme by concrete manufacturer limits with molecule.Make mixture heated to 95 ℃, with all preformed dsDNA complex that dissociate, other lower temperatures that slowly cool to room temperature subsequently or limited by test kit manufacturer are to allow dna molecular annealing.Recently the complex of Xing Chenging slowly is heated to 95 ℃ subsequently, and continuous data is collected when following about the fluorescence volume that produced by reaction.The dsDNA amount that exists in fluorescence intensity and the reaction is inversely proportional to.Data can be used the PCR in real time instrument compatible with test kit, and (Lewes UK) collects for for example ABI ' s StepOne Plus Real Time PCR System or LightTyper instrument, Roche Diagnostics.

Use appropriate software (for example LightTyper(Roche) or SDS Dissociation Curve, ABI), make up the peak that unwinds by mark and draw the negative derivant of fluorescence with regard to temperature (on the y axle-d(fluorescence)/dT) at temperature (x axle).Analytical data is to identify the temperature of the fast transition from the dsDNA complex to single chain molecule.This temperature is called as Tm, and is directly proportional with interaction strength between 2 kinds of molecules.Usually, Tm will be above 40 ℃.

Embodiment 2: the adjusting of lipid transfer and metabolic gene polynucleotide

Handle the 518A2 cell with antisense oligonucleotide:

Make the Center available from Albert Einstein-Montefiore Cancer, the 518A2 cell of NY is at growth medium (MEM/EBSS(Hyclone catalog number (Cat.No.) SH30024 or Mediatech catalog number (Cat.No.) MT-10-010-CV)+10%FBS(Mediatech catalog number (Cat.No.) MT35-011-CV)+penicillin/streptomycin (Mediatech catalog number (Cat.No.) MT30-002-CI)) at 37 ℃ and 5%CO ₂Following growth.Testing the previous day, make cell with 1.5 * 10 ⁵The density of/ml repaves in 6 orifice plates, and at 37 ℃ and 5%CO ₂Under hatch.Testing the same day, the culture medium in 6 orifice plates is replaced with fresh growth medium.All antisense oligonucleotides are diluted to the concentration of 20 μ M.Make this solution of 2 μ l and 400 μ l Opti-MEM culture medium (Gibco catalog number (Cat.No.) 31985-070) and 4 μ l Lipofectamine 2000 (Invitrogen catalog number (Cat.No.) 11668019) incubated at room 20 minutes, and be applied to have each hole of 6 orifice plates of 518A2 cell.Comprise that 2 μ l water replace the similar mixture of oligonucleotide solution to be used to simulate the transfection contrast.At 37 ℃ and 5%CO ₂Under hatch 3-18 hour after, change culture medium into fresh growth medium.After adding antisense oligonucleotide 48 hours, remove culture medium, and use SV Total RNA Isolation System(catalog number (Cat.No.) Z3105 from Promega) or, from cell, extract RNA according to the description of manufacturer from the RNeasy Total RNA Isolation test kit (catalog number (Cat.No.) 74181) of Qiagen.600 ng RNA add are used in the reverse transcription reaction that Verso cDNA test kit (catalog number (Cat.No.) AB1453B) or High Capacity cDNA Reverse Transcription Kit (catalog number (Cat.No.) 4368813) from Thermo Scientific carry out, described in the scheme of manufacturer.CDNA from this reverse transcription reaction is used for by PCR in real time monitoring gene expression, it uses ABI Taqman Gene Expression Mix(catalog number (Cat.No.) 4369510) and by primer/probe (Applied Biosystems Taqman Gene Expression Assay of ABI design, Applied Biosystems Inc., Foster City CA).Use following PCR circulation: use 50 ℃ of StepOne Plus Real Time PCR Machine (Applied Biosystems) 2 minutes, 95 ℃ 10 minutes, 40 circulations (95 ℃ 15 seconds, 60 ℃ 1 minute).

Based on handling and simulate the difference in 18S standardization dCt value between the transfection sample, calculating multiple in gene expression after handling with antisense oligonucleotide changes.

The result

PCR in real time result shows, 48 h after using at a processing among the siRNAs of ABCA1 antisense AK311445 design, and the level of ABCA1 mRNA significantly increases (Figure 1A) in the 518A2 cell.

PCR in real time result shows, 48 h after using at 6 processing in the oligomer of ABCA1 antisense AK311445 design, and the level of ABCA1 mRNA significantly increases (Figure 1B) in the 518A2 cell.

Handle the 3T3 cell with antisense oligonucleotide:

Make 3T3 cell from ATCC (catalog number (Cat.No.) CRL-1658) at growth medium (MEM/EBSS(Hyclone catalog number (Cat.No.) SH30024 or Mediatech catalog number (Cat.No.) MT-10-010-CV)+10%FBS(Mediatech catalog number (Cat.No.) MT35-011-CV)+penicillin/streptomycin (Mediatech catalog number (Cat.No.) MT30-002-CI)) at 37 ℃ and 5%CO ₂Following growth.Testing the previous day, make cell with 1.5 * 10 ⁵The density of/ml repaves in 6 orifice plates, and at 37 ℃ and 5%CO ₂Under hatch.Testing the same day, the culture medium in 6 orifice plates is replaced with fresh growth medium.All antisense oligonucleotides are diluted to the concentration of 20 μ M.Make this solution of 2 μ l and 400 μ l Opti-MEM culture medium (Gibco catalog number (Cat.No.) 31985-070) and 4 μ l Lipofectamine 2000 (Invitrogen catalog number (Cat.No.) 11668019) incubated at room 20 minutes, and be applied to have each hole of 6 orifice plates of 3T3 cell.Comprise that 2 μ l water replace the similar mixture of oligonucleotide solution to be used to simulate the transfection contrast.At 37 ℃ and 5%CO ₂Under hatch 3-18 hour after, change culture medium into fresh growth medium.After adding antisense oligonucleotide 48 hours, remove culture medium, and use SV Total RNA Isolation System(catalog number (Cat.No.) Z3105 from Promega) or, from cell, extract RNA according to the description of manufacturer from the RNeasy Total RNA Isolation test kit (catalog number (Cat.No.) 74181) of Qiagen.600 ng RNA add are used in the reverse transcription reaction that Verso cDNA test kit (catalog number (Cat.No.) AB1453B) or High Capacity cDNA Reverse Transcription Kit (catalog number (Cat.No.) 4368813) from Thermo Scientific carry out, described in the scheme of manufacturer.CDNA from this reverse transcription reaction is used for by PCR in real time monitoring gene expression, it uses ABI Taqman Gene Expression Mix(catalog number (Cat.No.) 4369510) and by primer/probe (Applied Biosystems Taqman Gene Expression Assay of ABI design, Applied Biosystems Inc., Foster City CA).Use following PCR circulation: use 50 ℃ of StepOne Plus Real Time PCR Machine (Applied Biosystems) 2 minutes, 95 ℃ 10 minutes, 40 circulations (95 ℃ 15 seconds, 60 ℃ 1 minute).

Based on handling and simulate the difference in 18S standardization dCt value between the transfection sample, calculating multiple in gene expression after handling with antisense oligonucleotide changes.

The result

PCR in real time result shows, 48 h after using at 3 processing in the oligomer of mice ABCA1 antisense BF133827 design, and the level of ABCA1 mRNA significantly increases (Fig. 1 C) in the 3T3 cell.

PCR in real time result shows that at oligomer processing back 48 h that use at LRP1 antisense DC401271 and AW544265, the level of LRP1 mRNA significantly increases (Fig. 1 J) in the 3T3 cell.

Handle the HepG2 cell with antisense oligonucleotide

Method 1: handle the HepG2 cell with naked antisense oligonucleotide:

Make HepG2 cell (catalog number (Cat.No.) HB-8065) at growth medium (MEM/EBSS(Hyclone catalog number (Cat.No.) SH30024 or Mediatech catalog number (Cat.No.) MT-10-010-CV)+10%FBS(Mediatech catalog number (Cat.No.) MT35-011-CV from ATCC)+penicillin/streptomycin (Mediatech catalog number (Cat.No.) MT30-002-CI)) at 37 ℃ and 5%CO ₂Following growth.Testing the previous day, make cell with 0.5 * 10 ⁴The density of/ml repaves in 6 orifice plates, and at 37 ℃ and 5%CO ₂Under hatch.Testing the same day, the culture medium in 6 orifice plates is replaced with 1.5 ml/ hole fresh growth medium.All antisense oligonucleotides are diluted to the concentration of 20 μ M in water.This solution of 2 μ l is mixed with 400 μ l fresh growth medium, and be applied to have each hole of 6 orifice plates of HepG2 cell.Comprise that 2 μ l water replace the similar mixture of oligonucleotide solution to be used for the simulation process contrast.At 37 ℃ and 5%CO ₂Under hatch 3-18 hour after, change culture medium into fresh growth medium.After adding antisense oligonucleotide 72 hours, the cell rechallenge that makes as noted before.After the administration second time 48-72 hour, remove culture medium, and use SV Total RNA Isolation System(catalog number (Cat.No.) Z3105 from Promega) or, from cell, extract RNA according to the description of manufacturer from the RNeasy Total RNA Isolation test kit (catalog number (Cat.No.) 74181) of Qiagen.600 ng RNA are added in the reverse transcription reaction of use from Verso cDNA test kit (catalog number (Cat.No.) AB1453B) execution of Thermo Scientific, described in the scheme of manufacturer.CDNA from this reverse transcription reaction is used for by PCR in real time monitoring gene expression, it uses ABI Taqman Gene Expression Mix(catalog number (Cat.No.) 4369510) and by the primer/probe (Applied Biosystems Inc., Foster City CA) of ABI design.Use following PCR circulation: use 50 ℃ of Mx4000 thermal cycler (Stratagene) 2 minutes, 95 ℃ 10 minutes, 40 circulations (95 ℃ 15 seconds, 60 ℃ 1 minute).Based on handling and simulate the difference in 18S standardization dCt value between the transfection sample, calculating multiple in gene expression after handling with antisense oligonucleotide changes.

Method 2: handle the HepG2 cell with antisense oligonucleotide:

Make HepG2 cell (catalog number (Cat.No.) HB-8065) at growth medium (MEM/EBSS(Hyclone catalog number (Cat.No.) SH30024 or Mediatech catalog number (Cat.No.) MT-10-010-CV)+10%FBS(Mediatech catalog number (Cat.No.) MT35-011-CV from ATCC)+penicillin/streptomycin (Mediatech catalog number (Cat.No.) MT30-002-CI)) at 37 ℃ and 5%CO ₂Following growth.Testing the previous day, make cell with 1.5 * 10 ⁵The density of/ml repaves in 6 orifice plates, and at 37 ℃ and 5%CO ₂Under hatch.Testing the same day, the culture medium in 6 orifice plates is replaced with fresh growth medium.All antisense oligonucleotides are diluted to the concentration of 20 μ M.Make this solution of 2 μ l and 400 μ l Opti-MEM culture medium (Gibco catalog number (Cat.No.) 31985-070) and 4 μ l Lipofectamine 2000 (Invitrogen catalog number (Cat.No.) 11668019) incubated at room 20 minutes, and be applied to have each hole of 6 orifice plates of HepG2 cell.Comprise that 2 μ l water replace the similar mixture of oligonucleotide solution to be used to simulate the transfection contrast.At 37 ℃ and 5%CO ₂Under hatch 3-18 hour after, change culture medium into fresh growth medium.After adding antisense oligonucleotide 48 hours, remove culture medium, and use SV Total RNA Isolation System(catalog number (Cat.No.) Z3105 from Promega) or, from cell, extract RNA according to the description of manufacturer from the RNeasy Total RNA Isolation test kit (catalog number (Cat.No.) 74181) of Qiagen.600 ng RNA are added in the reverse transcription reaction of use from Verso cDNA test kit (catalog number (Cat.No.) AB1453B) execution of Thermo Scientific, described in the scheme of manufacturer.CDNA from this reverse transcription reaction is used for by PCR in real time monitoring gene expression, it uses ABI Taqman Gene Expression Mix(catalog number (Cat.No.) 4369510) and by the primer/probe (Applied Biosystems Inc., Foster City CA) of ABI design.Use following PCR circulation: use 50 ℃ of Mx4000 thermal cycler (Stratagene) 2 minutes, 95 ℃ 10 minutes, 40 circulations (95 ℃ 15 seconds, 60 ℃ 1 minute).Based on handling and simulate the difference in 18S standardization dCt value between the transfection sample, calculating multiple in gene expression after handling with antisense oligonucleotide changes.

The result

PCR in real time result shows, 48 h after using at two processing in the oligomer of LCAT antisense Hs.668679 design, and the level of LCAT mRNA significantly increases (Fig. 1 E) in the HepG2 cell.

PCR in real time result shows, 48 h after using at a processing in the oligomer of LCAT antisense Hs.668679 design, and the level of LCAT mRNA significantly increases (Fig. 1 F) in the HepG2 cell.

PCR in real time result shows that at oligomer processing back 48 h that use at LRP1 antisense DC401271, the level of LRP1 mRNA significantly increases (Fig. 1 H) in the HepG2 cell.

PCR in real time result shows that at antisense scant polymer processing back 48 h that use at LDLR antisense sherflor.aApr07, the level of LDLr mRNA significantly increases in the HepG2 cell.Oligomer (CUR-1059-CUR-1063) at LDLr antisense bloflor.aApr07 design does not improve LDLr level (Fig. 1 K and 1L).

PCR in real time result shows, 48 h after using at three processing in the antisense scant polymer of APOE antisense Hs.626623 design, and the level of APOE mRNA significantly increases in the HepG2 cell.Oligomer at APOE4 antisense Hs.714236 design does not significantly improve APOE mRNA (figure M).

PCR in real time result shows, is using at some processing back 48 h in the antisense scant polymer of ApoA1 antisense DA327409ext, and the level of ApoA1 mRNA significantly increases (Fig. 1 N to Fig. 1 P) in the HepG2 cell.

PCR in real time result shows, and compares, after handling the HepG2 cell with naked LNA or thiophosphate oligonucleotide during 7 days, at the last figure of ApoA1 mRNA() and ApoA1 natural antisense DA327409ext RNA(figure below) in multiple variation (scheming Q).

PCR in real time result shows, after handling the HepG2 cell with the LNA oligonucleotide, orange of ApoA1 mRNA() and ApoA1 natural antisense DA327409ext RNA(blue bar) in multiple change (scheming R).

Handle with antisense oligonucleotideHek293 Cell:

Make Hek293 cell (catalog number (Cat.No.) CRL-1573) at growth medium (MEM/EBSS(Hyclone catalog number (Cat.No.) SH30024 or Mediatech catalog number (Cat.No.) MT-10-010-CV)+10%FBS(Mediatech catalog number (Cat.No.) MT35-011-CV from ATCC)+penicillin/streptomycin (Mediatech catalog number (Cat.No.) MT30-002-CI)) at 37 ℃ and 5%CO ₂Following growth.Testing the previous day, make cell with 1.5 * 10 ⁵The density of/ml repaves in 6 orifice plates, and at 37 ℃ and 5%CO ₂Under hatch.Testing the same day, the culture medium in 6 orifice plates is replaced with fresh growth medium.All antisense oligonucleotides are diluted to the concentration of 20 μ M.Make this solution of 2 μ l and 400 μ l Opti-MEM culture medium (Gibco catalog number (Cat.No.) 31985-070) and 4 μ l Lipofectamine 2000 (Invitrogen catalog number (Cat.No.) 11668019) incubated at room 20 minutes, and be applied to have each hole of 6 orifice plates of Hek293 cell.Comprise that 2 μ l water replace the similar mixture of oligonucleotide solution to be used to simulate the transfection contrast.At 37 ℃ and 5%CO ₂Under hatch 3-18 hour after, change culture medium into fresh growth medium.After adding antisense oligonucleotide 48 hours, remove culture medium, and use SV Total RNA Isolation System(catalog number (Cat.No.) Z3105 from Promega) or, from cell, extract RNA according to the description of manufacturer from the RNeasy Total RNA Isolation test kit (catalog number (Cat.No.) 74181) of Qiagen.600 ng RNA add are used in the reverse transcription reaction that Verso cDNA test kit (catalog number (Cat.No.) AB1453B) or High Capacity cDNA Reverse Transcription Kit (catalog number (Cat.No.) 4368813) from Thermo Scientific carry out, described in the scheme of manufacturer.CDNA from this reverse transcription reaction is used for by PCR in real time monitoring gene expression, it uses ABI Taqman Gene Expression Mix(catalog number (Cat.No.) 4369510) and by primer/probe (Applied Biosystems Taqman Gene Expression Assay of ABI design, Applied Biosystems Inc., Foster City CA).Use following PCR circulation: use Mx4000 thermal cycler (Stratagene) or StepOne Plus Real Time PCR Machine (Applied Biosystems) 50 ℃ 2 minutes, 95 ℃ 10 minutes, 40 circulations (95 ℃ 15 seconds, 60 ℃ 1 minute).

Based on handling and simulate the difference in 18S standardization dCt value between the transfection sample, calculating multiple in gene expression after handling with antisense oligonucleotide changes.

The result:

PCR in real time result shows, 48 h after using at three processing in the oligomer of LCAT antisense Hs.668679 design, and the level of LCAT mRNA significantly increases (Fig. 1 D) in the Hek293 cell.

Handle Vero 76 cells with antisense oligonucleotide

Make Vero 76 cells (catalog number (Cat.No.) CRL-1587) from ATCC at growth medium (MEM/EBSS(Hyclone catalog number (Cat.No.) SH30024 or Mediatech catalog number (Cat.No.) MT-10-010-CV)+10%FBS(Mediatech catalog number (Cat.No.) MT35-011-CV)+penicillin/streptomycin (Mediatech catalog number (Cat.No.) MT30-002-CI)) at 37 ℃ and 5%CO ₂Following growth.Testing the previous day, make cell with 1.5 * 10 ⁵The density of/ml repaves in 6 orifice plates, and at 37 ℃ and 5%CO ₂Under hatch.Testing the same day, the culture medium in 6 orifice plates is replaced with fresh growth medium.All antisense oligonucleotides are diluted to the concentration of 20 μ M.Make this solution of 2 μ l and 400 μ l Opti-MEM culture medium (Gibco catalog number (Cat.No.) 31985-070) and 4 μ l Lipofectamine 2000 (Invitrogen catalog number (Cat.No.) 11668019) incubated at room 20 minutes, and be applied to have each hole of 6 orifice plates of Vero 76 cells.Comprise that 2 μ l water replace the similar mixture of oligonucleotide solution to be used to simulate the transfection contrast.At 37 ℃ and 5%CO ₂Under hatch 3-18 hour after, change culture medium into fresh growth medium.After adding antisense oligonucleotide 48 hours, remove culture medium, and use SV Total RNA Isolation System(catalog number (Cat.No.) Z3105 from Promega) or, from cell, extract RNA according to the description of manufacturer from the RNeasy Total RNA Isolation test kit (catalog number (Cat.No.) 74181) of Qiagen.600 ng RNA add are used in the reverse transcription reaction that Verso cDNA test kit (catalog number (Cat.No.) AB1453B) or High Capacity cDNA Reverse Transcription Kit (catalog number (Cat.No.) 4368813) from Thermo Scientific carry out, described in the scheme of manufacturer.CDNA from this reverse transcription reaction is used for by PCR in real time monitoring gene expression, it uses ABI Taqman Gene Expression Mix(catalog number (Cat.No.) 4369510) and by primer/probe (Applied Biosystems Taqman Gene Expression Assay of ABI design, Applied Biosystems Inc., Foster City CA).Use following PCR circulation: use Mx4000 thermal cycler (Stratagene) or StepOne Plus Real Time PCR Machine (Applied Biosystems) 50 ℃ 2 minutes, 95 ℃ 10 minutes, 40 circulations (95 ℃ 15 seconds, 60 ℃ 1 minute).

Based on handling and simulate the difference in 18S standardization dCt value between the transfection sample, calculating multiple in gene expression after handling with antisense oligonucleotide changes.

The result

PCR in real time result shows, 48 h after using at a processing in the oligomer of LCAT antisense Hs.668679 design, and the level of LCAT mRNA significantly increases (Fig. 1 G) in the Vero cell.

PCR in real time result shows that at oligomer processing back 48 h that use at LRP1 antisense DC401271 and Hs.711951, the level of LRP1 mRNA significantly increases (Fig. 1 I) in the Vero cell.

The detector probe that is used for Applied Biosystems Gene Expression Assays:

ABCA1:Hs00194045_m1 (people), Mm01350760_m1 (mice)

LCAT: Hs00173415_m1

LRP1:Hs00233856_m1 (people), Mm00464608_m1 (mice)

LDLR: Hs00181192_m1

ApoE: Hs00171168_m1

ApoA1:Hs00163641_m1,18S catalog number (Cat.No.) 4319413E

Analysis to ApoA1 antisense DA327409ext custom design:

FAM, labelling: TTTGGATCTGGACGACTTC (SEQ ID NO:275).

Embodiment 3: the adjusting that lipid transfer and metabolic gene are expressed

Material and method

With the arbitrary processing cell in the following method:

Method 1: handle the HepG2 cell with naked antisense oligonucleotide:

Make the HepG2 cell at MEM/EBSS(Hyclone catalog number (Cat.No.) SH30024)+10%FBS+penicillin/streptomycin at 37 ℃ and 5%CO ₂Following growth.Testing the previous day, make cell with 1.5 * 10 ⁴The density of/ml repaves in 6 orifice plates, and places 37 ℃ and 5%CO ₂Down.Testing the same day, the culture medium in 6 orifice plates is replaced with fresh MEM/EBSS+10%FBS.Be diluted to the concentration of 20 μ M by all antisense oligonucleotides of IDT manufacturing.This solution of 2 μ l is mixed with 400 μ l Opti-MEM culture medium (Gibco catalog number (Cat.No.) 31985-070), and be applied to have each hole of 6 orifice plates of HepG2 cell.Comprise that 2 μ l water replace the similar mixture of oligonucleotide solution to be used to simulate the transfection contrast.After adding antisense oligonucleotide 72 hours, remove culture medium, and as above-mentioned repeat administration program.

48-72 h behind the repeat administration RNA, use is from the SV Total RNA Isolation System(catalog number (Cat.No.) Z3105 of Promega) or, from cell, extract RNA according to the description of manufacturer from the RNeasy Total RNA Isolation test kit (catalog number (Cat.No.) 74181) of Qiagen.600 ng RNA are added in the reverse transcription reaction of use from Verso cDNA test kit (catalog number (Cat.No.) AB1453B) execution of Thermo Scientific, described in the scheme of manufacturer.CDNA from this reverse transcription reaction is used for by PCR in real time monitoring gene expression, it uses ABI Taqman Gene Expression Mix(catalog number (Cat.No.) 4369510) and by the primer/probe (Applied Biosystems Inc., Foster City CA) of ABI design.Use following PCR circulation: use 50 ℃ of Mx4000 thermal cycler (Stratagene) 2 minutes, 95 ℃ 10 minutes, 40 circulations (95 ℃ 15 seconds, 60 ℃ 1 minute).

Based on handling and simulate the difference in 18S standardization dCt value between the transfection sample, calculating multiple in gene expression after handling with antisense oligonucleotide changes.

The primer and the probe that are used for the custom-designed Taqman mensuration of ApoA1 natural antisense DA327409ext.The deoxyribonucleotide of capitalization indication unmodified:

Probe sequence (FAM, labelling) TTTGGATCTGGACGACTTC (SEQ ID NO:275)

Forward primer sequence C TCCTCCTGCCACTTCTTCTG (SEQ ID NO:276)

Reverse primer sequence C TGGTGGATGAAGAAGGTTTGC (SEQ ID NO:277).

Method 2: handle the HepG2 cell with antisense oligonucleotide:

Make HepG2 cell (catalog number (Cat.No.) HB-8065) at growth medium (MEM/EBSS(Hyclone catalog number (Cat.No.) SH30024 or Mediatech catalog number (Cat.No.) MT-10-010-CV)+10%FBS(Mediatech catalog number (Cat.No.) MT35-011-CV from ATCC)+penicillin/streptomycin (Mediatech catalog number (Cat.No.) MT30-002-CI)) at 37 ℃ and 5%CO ₂Following growth.Testing the previous day, make cell with 1.5 * 10 ⁵The density of/ml repaves in 6 orifice plates, and at 37 ℃ and 5%CO ₂Under hatch.Testing the same day, the culture medium in 6 orifice plates is replaced with fresh growth medium.All antisense oligonucleotides are diluted to the concentration of 20 μ M.Make this solution of 2 μ l and 400 μ l Opti-MEM culture medium (Gibco catalog number (Cat.No.) 31985-070) and 4 μ l Lipofectamine 2000(Invitrogen catalog number (Cat.No.)s 11668019) at room temperature hatch 20 minutes, and be applied to have each hole of 6 orifice plates of HepG2 cell.Comprise that 2 μ l water replace the similar mixture of oligonucleotide solution to be used to simulate the transfection contrast.At 37 ℃ and 5%CO ₂Under hatch 3-18 hour after, change culture medium into fresh growth medium.After adding antisense oligonucleotide 48 hours, remove culture medium, and use SV Total RNA Isolation System(catalog number (Cat.No.) Z3105 from Promega) or, from cell, extract RNA according to the description of manufacturer from the RNeasy Total RNA Isolation test kit (catalog number (Cat.No.) 74181) of Qiagen.

600 ng RNA are added in the reverse transcription reaction of use from Verso cDNA test kit (catalog number (Cat.No.) AB1453B) execution of Thermo Scientific, described in the scheme of manufacturer.CDNA from this reverse transcription reaction is used for by PCR in real time monitoring gene expression, use ABI Taqman Gene Expression Mix(catalog number (Cat.No.) 4369510) and by the primer/probe (Applied Biosystems Inc., Foster City CA) of ABI design.Use following PCR circulation: use 50 ℃ of Mx4000 thermal cycler (Stratagene) 2 minutes, 95 ℃ 10 minutes, 40 circulations (95 ℃ 15 seconds, 60 ℃ 1 minute).Based on handling and simulate the difference in 18S standardization dCt value between the transfection sample, calculating multiple in gene expression after handling with antisense oligonucleotide changes.

The primer and the probe that are used for the custom-designed Taqman mensuration of ApoA1 natural antisense DA327409ext.The deoxyribonucleotide of capitalization indication unmodified:

Probe sequence (FAM, labelling) TTTGGATCTGGACGACTTC (SEQ ID NO:275)

Forward primer sequence C TCCTCCTGCCACTTCTTCTG (SEQ ID NO:276)

Reverse primer sequence C TGGTGGATGAAGAAGGTTTGC (SEQ ID NO:277).

The hepatocellular processing of former generation monkey

By RxGen Inc. former generation monkey hepatocyte introducing is cultivated, and at 6 orifice plate middle berth flat boards.They are following handles with oligonucleotide.Culture medium in 6 orifice plates is replaced with by William ' s Medium E(Sigma catalog number (Cat.No.) W4128) fresh growth medium formed, it is supplemented with 5%FBS, 50 U/ml penicillins and 50 ug/ml streptomycins, 4 ug/ml insulins, 1 uM dexamethasone, 10 ug/ml fungins (Fungin) (InVivogen, San Diego CA).All antisense oligonucleotides are diluted to the concentration of 20 μ M.Make this solution of 2 μ l and 400 μ l Opti-MEM culture medium (Gibco catalog number (Cat.No.) 31985-070) and 4 μ l Lipofectamine 2000(Invitrogen catalog number (Cat.No.)s 11668019) at room temperature hatch 20 minutes, and be applied to have each hole of 6 orifice plates of cell.Comprise that 2 μ l water replace the similar mixture of oligonucleotide solution to be used to simulate the transfection contrast.At 37 ℃ and 5%CO ₂Under hatch 3-18 hour after, change culture medium into fresh growth medium.After adding antisense oligonucleotide 48 hours, remove culture medium, and use SV Total RNA Isolation System(catalog number (Cat.No.) Z3105 from Promega) or, from cell, extract RNA according to the description of manufacturer from the RNeasy Total RNA Isolation test kit (catalog number (Cat.No.) 74181) of Qiagen.

600 ng RNA are added in the reverse transcription reaction of use from Verso cDNA test kit (catalog number (Cat.No.) AB1453B) execution of Thermo Scientific, described in the scheme of manufacturer.CDNA from this reverse transcription reaction is used for by PCR in real time monitoring gene expression, use ABI Taqman Gene Expression Mix(catalog number (Cat.No.) 4369510) and by the primer/probe (Applied Biosystems Inc., Foster City CA) of ABI design.Use following PCR circulation: use 50 ℃ of Mx4000 thermal cycler (Stratagene) 2 minutes, 95 ℃ 10 minutes, 40 circulations (95 ℃ 15 seconds, 60 ℃ 1 minute).Based on handling and simulate the difference in 18S standardization dCt value between the transfection sample, calculating multiple in gene expression after handling with antisense oligonucleotide changes.

According to the description of manufacturer, use MabTech Inc. ApoA1 ELISA test kit catalog number (Cat.No.) 3710-11-6 to carry out ELISA

The results are shown among Fig. 1 Q to Fig. 1 T.Figure Q show as by the last figure of detected ApoA1 mRNA() and ApoA1 antisense DA327409ext RNA(figure below) the measurement amount, the 2 kinds of oligonucleotide and the LNA oligonucleotide that have the thiophosphate main chain and be internucleotide linkage are effective in the adjusting expression of target gene.Figure R is presented at and uses at orange of the ApoA1 mRNA(in the acid-treated HepG2 cell of oligonucleoside of DA327409ext design) and ApoA1 antisense DA327409ext RNA(blue bar) level.Figure S is presented at and uses at ApoA1 mRNA(figure below in the acid-treated HepG2 culture of oligonucleoside of DA327409ext design) and the dose dependent rise of protein (last figure).Figure T be presented at use handle at the oligonucleotide of DA327409ext design after, the rise of ApoA1 mRNA in former generation cercopithecus aethiops hepatocyte.

Embodiment 4: the effect of CUR-962 and acting duration research in cercopithecus aethiops

The purpose of this research is that the antisense of assessment and more inharmonious non-encoding antisense sequence is knocked down effect, and described inharmonious non-encoding antisense sequence is used the back at intravenous and regulated lipid transfer and metabolic gene in the non-human primates model.Be designed to suppress the antisense oligonucleotide test article called after CUR-962 that APOA1 regulates sequence.

CUR-962：+G*+C*T* A*G*T* C*T*G* +T*+T*+G（SEQ ID NO：278）。

The CUR-963(contrast) :+G*+T*C* T*G*A* T*G*G*+A*+G*+A(SEQ ID NO:279).

Regulating test instructs

This research is according to the toxicology principle of generally acknowledging and abide by International Conference of Harmonization(ICH) Harmonized Tripartite Guidelines(Non-Clinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals ICH M3(m), 2000 November 9) and It is generally accepted that the operation that is used for the treatment of the agent test designs.

Test and contrast article

The tester product are identified and preparation

Test article CUR-962 is chemically stable antisense oligonucleotide.Being used for the carrier that intravenous sends is phosphate buffered saline (PBS) (PBS).

Carrier characterizes

For the PBS carrier, composition, lot number, expiry date and storage requirement (temperature and light/dark) derive from supplier.

Test article storage and processing

Test substances and carrier are stored according to the generally acknowledged storage requirement that is correspondingly provided by sponsor and manufacturer.

The analysis of test article preparation

The sample of test article preparation is used for freezing preservation concentration, stability and the homogeneity of analytical test substance preparation.

Test principle

Primates is can accept suitable non-Rodents species as the potential hazard indicator for administrative authority, and is obtainable about its extensive background information.Cercopithecus aethiops is the clinical correlation model of height of various human physiology and morbid state particularly.

The intravenous route of administration is corresponding with possible human therapy approach.The dosage of test article is found the result of research based on the dosage of the similar compound of before carrying out in cercopithecus aethiops.

Select cercopithecus aethiops as the primates of selecting, guard, in primates, have 100% homology because the target sequence of test article is crossed over species.In addition, test substances is a synthetic oligonucleotide.Therefore, administration allows the good assessment of these compound efficacy in primates, and described chemical compound will be than more be reflected in the visible picked-up of possibility among the people in any other species.

Animal

Species: titi (Chlorocebus sabaeus), non-human primates.

Kind:St. Kitts native country cercopithecus aethiops.

The source:RxGen, Lower Bourryeau, St. Kitts, West Indies.

The expectation age:Test animal is grown up.

Desired weight:The about 3-4 kg of monkey weight.Actual range can change but will be recorded in the data.

Sex:Test animal is adult female.

The animal number: Screen 10 animals to guarantee to be suitable for adding the evaluation of 8 animals in the research.

The research number:Female: 8.

Demonstration about the research number

This research design is for using the animal of minimum possibility number, and is consistent with the previous research that the main target and the general of this class oligonucleotide in these species of the therapeutic efficiency of assessment test article in cercopithecus aethiops are used.

The animal specification

Adopt 10 adult cercopithecus aethiopses of weight range 3 – 4 kg under study for action.Monkey is the adults that is used for medicine first that humanity is caught from the wild population that inhabits the island.The monkey that is hunted down is handled with anthelmintic (antihelminthics), loads to eliminate any possible celiosite, and quarantines 4 weeks of bottom line before adding screening with regard to research.Be hunted down age of monkey is estimated by size and tooth-shape structure, get rid of senior animal from studies.Before research adds, every monkey is carried out clinical examination, comprise and moving and dexterous assessment.Obtain blood sample and be sent to Antech Diagnostics(Memphis, TN), be used for comprehensive clinical chemistry and complete blood count and fat spectrum (referring to sections 9.2 and 319567928) about specification.As by with the comparative measurements of in St. Kitts colony, determining normal range, from research, get rid of monkey with unusual laboratory evaluation for monkey.In order to identify 8 monkeys of satisfying this standard, screen 10 monkeys, screen other animal as required.Before research beginning, the monkey of selecting is transferred to indivedual cages, to adapt to one period in week of individual breeding.Only think that the animal that is suitable for testing will add research.Reality (or estimation) age and weight range when the research beginning will describe in detail in initial data and final report (FR).

Animal health and welfare

Follow the animal welfare of highest standard and adhere to guidance by the St. Kitts Ministry of Agriculture (Department of Agriculture) and U.S. sanitary and public service portion (U.S. Department of Health and Human Services) regulation.All researchs will and be used for management of laboratory animal and but all application practice standards of raising are carried out according to these requirements.But about all application standards of veterinary management, operation and examination as comprising in NIH the care of animal and the instruction (Guide for the Care and Use of Animals).St. the Kitts facility keeps as the zooscopy committee by examination scheme that instructs requirement and inspection facility.Foundation has the approval of being submitted to by laboratory animal welfare office (Office of Laboratory Animal Welfare) and guarantees, as by instructing the biomedical foundation (Biomedical Foundation) of #A4384-01(Axion WARF (Research Foundation)/St. Kitts) require.Do not exist by specified special non-human primates veterinary's organize content and the biohazard content of researching and proposing in this research.

Raise and environment

In order to allow to detect any processing relevant clinical sign, the animal individual breeding is when putting to death before operation and after the operation.Indivedual cages are positioned at wherein primates building fully by ambient light illumination, recommend in instructing as U.S. D.H.H.S, and this was about 12 hours of north latitude 17 degree: 12 little time-dark cycle.RxGen primates building is vented to outdoor fully.Other air flows through ceiling fan to be guaranteed, keeping 23-35 ℃ constant target temperature, as St. Kitts is annual.Measure 24 hours extreme and relative humiditys (this is also with uncontrolled) of temperature every day.In research process, cage regularly cleans.

Diet and water

Every animal provide about 90 gram/skies standard monkey food diet (TekLad, Madison, WI).The concrete trophic component of record diet.Water periodic analysis microorganism purity.About the pollutant standard of acceptable level in the supply of deposit diet and water respectively in analysis specification of determining by diet manufacturer and facility water assessment regularly.Water meets for proving can be accepted to be used for the people and consume all required standards.

Experimental design

Animal identification and randomization

By means of finishing distribution based on the stratified random operation of body weight and plasma cholesterol spectrum.Be dispensed to the group before and after, every animal is identified by tatooing on the abdominal part.Tatooing places on all colony animals, as the authentication method in conventional physical examination process.Draft the cage plan identifying individuality in interior raising, and indivedual monkey by with its respectively the markup tags that adheres to of cage further identify.

Group size, dosage and identification number

Animal is dispensed to 2 processed group, is made up of 4 monkeys in each group.According to the facility numbering system, provide special animal identification number to every monkey.This system by letter be subsequently 3 digital number for example Y032 identify every monkey uniquely.

Route of administration and frequency

In the time of send at intravenous the 1st, 3 and 5 day, animal by manual infusion through being administered once ~ 10 minute every day.Infusion rate will be 24 mL/kg/ hours.Before the administration operation and in the process, animal carries out calmness with ketamine and xylazine.Venous duct (the miniature venous transfusion device of Terumo, No. 20 pins, or similar suitable infusion set) is inserted in the saphena.Administration takes place between 8:00 and 10:00 a.m after animal is waken up in every monkey soon and on the feed.Described in hereinafter hematochemistry sections, only before each infusion, collect blood sample, with assessment plasma cholesterol and other lipid levels.Before the feed of blood sampling when 2 sub-samplings interval, so that the effect that diet is measured cholesterol drops to minimum.

Clinical observation

All visible signs of record therapeutic response when the every day administration.In addition, animal is checked at least once weekly with regard to the general situation of for example outer harmony in the exterior of physical attribute.

Body weight

Period is to write down body weight weekly at interval after the processing procedure neutralisation treatment.

Food consumption

The individual food consumption of non-quantitative.Yet, monitoring feed mode and the remarks of making any bigger change.

Mortality rate and sickness rate

To write down mortality rate and sickness rate.After lead study author and possible sponsor's monitoring scientist discusses, make any decision about too early execution.To implement postmortem to the animal of finding death or being killed too early, and collect liver, kidney, heart and spleen lung tissue and be used for histopathology.In the incident of putting to death too early, also will obtain blood sample when possible () and location parameter.After the steady job time, find dead animal with freeze overnight, and when begin next working day, carry out postmortem.If the animal situation needs to put to death too early, it will implement euthanasia by the pentobarbital sodium of intravenous overtreatment so.All researchs are subjected to animal using priciple (Principles for Use of Animals) control.RxGen abides by U.S. sanitary and public service portion primates facility standard by legal requiremnt, and this indication must be observed the seriousness level of being appointed as gentle operation in this research.

Clinical laboratory's research

Blood sample

Before processing, obtain 3 blood samples, to determine the plasma cholesterol baseline from all animals.After processing, collect blood sample and obtain via shallow table venipuncture.The volume of collecting when any one sampling time point is no more than 8 ml, and this represents about 4% total blood volume of Adult Monkey.

Animal when 2 baseline time points and in research time blood drawing in the 1st, 3,5,7,9,11,13 and 15 day, if follow recognize that disturbance continues so thereafter～collect weekly, until the plasma cholesterol standardization of organizing in 1 (APOA1).In the time of the 1st, 6 and 11 day, collect 8 milliliters of blood, to allow assessment clinical chemistry, lipodogramme and to solidify spectrum.When every other natural law, only collect 5 milliliters of blood that enough are used for clinical chemistry and lipodogramme.

Measure and the same day blood sample is divided into 3 parts carrying out chemistry and hematology.A sample collection in the plasma collection pipe that comprises 25 μ l heparin, and is marked with research numbering, dosage level, natural law, date, unique animal identification number.After separation, take out 1 milliliter of blood plasma to the aseptic frozen pipe that carries above-mentioned details, and suitably storage is used for blood chemical analysis until transporting.Take out a blood plasma aliquot (0.5 milliliter) to the aseptic frozen pipe that is marked with above-mentioned details, and suitably storage is used for plasma cholesterol distribution and lipodogramme analysis until transporting.Make other 1 milliliter and the quick-freezing of 0.5 milliliter of blood plasma aliquot and be stored in the liquid nitrogen, be used for possible other analysis to serve as the backup sample.

2 other whole blood aliquots (each 2.5 milliliters) are handled and labelling with acid citrate dextrose (ACD) anticoagulant, and are stored in 4 ℃ and measure until transporting solidifying with CBC of being used for hereinafter describing in detail.

Transport sample reaching in 24 hours, or under steady-state conditions, store and be used for transporting in definite suitable time in sampling.

Only when being considered to exceed the normal quality restriction, sampling method or assay method obtain repeat samples.Sample is got access in the labelling pipe.

The hematology

The all samples that the 1st, 6 and 11 day the time when detecting turbulent other day during any in these time points (and if) collects is measured complete blood count (CBC), prothrombin time, PTT, Fibrinogen and D-dimer.To 1 milliliter of whole blood assessment cytometry in comprising the vacuum test tube of EDTA, collecting.Spectrum is solidified in about 2.0 milliliters of blood execution of collecting in the vacuum test tube that comprises acid citrate dextrose (ACD) anticoagulant to be measured.

Hematochemistry

Glucose, blood urea nitrogen, kreatinin, gross protein, albumin, total bilirubin, alkali phosphatase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol, calcium, phosphorus, sodium, potassium, chloride, A/G ratio, BUN/ kreatinin (calculating), globulin (calculating), lipase, amylase, triglyceride, CPK, lactic acid dehydrogenase, γ glutamyl transferase (GGT), magnesium, T-CHOL LDL, VLDL, HDL, ApoA1, ApoA2, ApoB, ApoE, ApoLp(a).Each plasma sample is carried out metachemistry (superchemistries) and LDL and HDL to be measured.After assessment LDL and HDL data, measure selecting sample to carry out ApoA1.

About 1.0 mL blood plasma execution mensuration is used for metachemistry measures, and 0.5ml blood plasma execution mensuration is used for cholesterol distribution and lipid transfer and metabolic gene measurement.Collect other blood plasma aliquot and storage and be used for possible futures analysis.

Liver biopsy

When baseline and in the time of the 7th and 17 day, all monkeys are carried out the percutaneous liver biopsy.To adopt No. 14 biopsy needles (INRAD), to obtain 2 center biopsies (length ~ 1.0 cm) of and lobus sinister right from liver.Before branch more as described below, by the successful biopsy of visual examination confirmation of the biopsy samples on biopsy needle.

Merge sample and separate in the following manner subsequently.To bioptic half (~ 0.5 cm) immerse in the paraformaldehyde from of lobus sinister, and be used for section and be used for histopathology and original position analysis.Immediately each is separated bioptic all the other half and other 2 complete biopsies immerse comprise 2 mls RNAlater(Qiagen) the frozen pipe of labelling in, and, aspirate RNAlater and make sample cell quick-freezing in liquid nitrogen after this 4 ℃ of following overnight incubation.After in liquid nitrogen, transporting, adopt Trizol or TriReagent method to separate total RNA, have ~ the bioptic expectation yield in 40 μ g/1.0 cm, 14 g centers (for derived from from all 4 the bioptic merging in merging center RNA of single monkey altogether ~ 80-100 μ g, do not exist and preserve the component that is used for histopathology and original position).5 μ g RNA fraction are used for the real-time qPCR(TaqMan miRNA of target-specific to be measured, ABI).All the other RNA fraction are preserved and are used for possible full genomic expression analysis.

Handle fixing organization and be used for paraffin embedding.Section statining is used for H﹠E and reporting organization's pathology discovery under the naked eyes histology finds.All microscope slides that in this work, generate carry have research numbering, dosage level, the labelling of natural law, date, unique animal identification number.

Statistical analysis

Statistics

Execution is about the descriptive statistics of hematology, clinical chemistry and lipodogramme.Expression data is carried out suitable bioinformatic analysis.

Sample size

Carry out sample size mensuration based on use the previous experiment and the correlated variability that modify antisense oligonucleotide and generation clinical chemistry and lipodogramme change to cercopithecus aethiops.The experimenter's total number that is used for efficacy assessment is 20 addings (enrolled) animals, wherein 4 animal/processed group and 4 animals of screening in addition.

The result:

The results are shown among the following figure.Fig. 1 U: as (2 the left figure) that measures by PCR in real time and ELISA respectively, compare with baseline values, after handling at the oligonucleotide of ApoA1 antisense DA327409ext design with CUR-962, the last figure of ApoA1 mRNA(that in the monkey liver biopsy, increases) and protein (figure below) level.Be used in external demonstration the ApoA1 level is not had in the matched group of oligonucleotide (CUR-963,2 right figure) administration of effect after the identical time period, ApoA1 mRNA and protein level do not change.

Although the present invention illustrates and describes with regard to one or more realizations, after reading and understanding this description and accompanying drawing, those skilled in the art will expect of equal value the change and modification.In addition, though special characteristic of the present invention can with regard to unique one in several realizations, disclose, this category feature can with one or more other characteristics combination of other realizations, as can being required and favourable for any given or application-specific.

The summary of disclosure will allow the reader to determine the character of technology disclosure fast.This summary and understanding are provided, and it will be not used in explains or limits following claim scope or implication.

Sequence table

<110> CuRNA, Inc.

＜120〉by suppression therapy lipid transfer and metabolic gene relevant disease at lipid transfer and metabolic natural antisense transcript

<130> LTMG

<150> US61/175,930

<151> 2009-05-06

<150> US61/180,646

<151> 2009-05-22

<150> US61/248,212

<151> 2009-10-02

<150> US61/235,227

<151> 2009-08-19

<150> US61/176,267

<151> 2009-05-07

<160> 279

<170> PatentIn version 3.5

<210> 1

<211> 10412

<212> DNA

＜213〉homo sapiens

<300>

<308> NM_005502

<309> 2010-08-15

<313> (1)..(10412)

<400> 1

gtaattgcga gcgagagtga gtggggccgg gacccgcaga gccgagccga cccttctctc 60

ccgggctgcg gcagggcagg gcggggagct ccgcgcacca acagagccgg ttctcagggc 120

gctttgctcc ttgttttttc cccggttctg ttttctcccc ttctccggaa ggcttgtcaa 180

ggggtaggag aaagagacgc aaacacaaaa gtggaaaaca gttaatgacc agccacggcg 240

tccctgctgt gagctctggc cgctgccttc cagggctccc gagccacacg ctgggggtgc 300

tggctgaggg aacatggctt gttggcctca gctgaggttg ctgctgtgga agaacctcac 360

tttcagaaga agacaaacat gtcagctgct gctggaagtg gcctggcctc tatttatctt 420

cctgatcctg atctctgttc ggctgagcta cccaccctat gaacaacatg aatgccattt 480

tccaaataaa gccatgccct ctgcaggaac acttccttgg gttcagggga ttatctgtaa 540

tgccaacaac ccctgtttcc gttacccgac tcctggggag gctcccggag ttgttggaaa 600

ctttaacaaa tccattgtgg ctcgcctgtt ctcagatgct cggaggcttc ttttatacag 660

ccagaaagac accagcatga aggacatgcg caaagttctg agaacattac agcagatcaa 720

gaaatccagc tcaaacttga agcttcaaga tttcctggtg gacaatgaaa ccttctctgg 780

gttcctgtat cacaacctct ctctcccaaa gtctactgtg gacaagatgc tgagggctga 840

tgtcattctc cacaaggtat ttttgcaagg ctaccagtta catttgacaa gtctgtgcaa 900

tggatcaaaa tcagaagaga tgattcaact tggtgaccaa gaagtttctg agctttgtgg 960

cctaccaagg gagaaactgg ctgcagcaga gcgagtactt cgttccaaca tggacatcct 1020

gaagccaatc ctgagaacac taaactctac atctcccttc ccgagcaagg agctggctga 1080

agccacaaaa acattgctgc atagtcttgg gactctggcc caggagctgt tcagcatgag 1140

aagctggagt gacatgcgac aggaggtgat gtttctgacc aatgtgaaca gctccagctc 1200

ctccacccaa atctaccagg ctgtgtctcg tattgtctgc gggcatcccg agggaggggg 1260

gctgaagatc aagtctctca actggtatga ggacaacaac tacaaagccc tctttggagg 1320

caatggcact gaggaagatg ctgaaacctt ctatgacaac tctacaactc cttactgcaa 1380

tgatttgatg aagaatttgg agtctagtcc tctttcccgc attatctgga aagctctgaa 1440

gccgctgctc gttgggaaga tcctgtatac acctgacact ccagccacaa ggcaggtcat 1500

ggctgaggtg aacaagacct tccaggaact ggctgtgttc catgatctgg aaggcatgtg 1560

ggaggaactc agccccaaga tctggacctt catggagaac agccaagaaa tggaccttgt 1620

ccggatgctg ttggacagca gggacaatga ccacttttgg gaacagcagt tggatggctt 1680

agattggaca gcccaagaca tcgtggcgtt tttggccaag cacccagagg atgtccagtc 1740

cagtaatggt tctgtgtaca cctggagaga agctttcaac gagactaacc aggcaatccg 1800

gaccatatct cgcttcatgg agtgtgtcaa cctgaacaag ctagaaccca tagcaacaga 1860

agtctggctc atcaacaagt ccatggagct gctggatgag aggaagttct gggctggtat 1920

tgtgttcact ggaattactc caggcagcat tgagctgccc catcatgtca agtacaagat 1980

ccgaatggac attgacaatg tggagaggac aaataaaatc aaggatgggt actgggaccc 2040

tggtcctcga gctgacccct ttgaggacat gcggtacgtc tgggggggct tcgcctactt 2100

gcaggatgtg gtggagcagg caatcatcag ggtgctgacg ggcaccgaga agaaaactgg 2160

tgtctatatg caacagatgc cctatccctg ttacgttgat gacatctttc tgcgggtgat 2220

gagccggtca atgcccctct tcatgacgct ggcctggatt tactcagtgg ctgtgatcat 2280

caagggcatc gtgtatgaga aggaggcacg gctgaaagag accatgcgga tcatgggcct 2340

ggacaacagc atcctctggt ttagctggtt cattagtagc ctcattcctc ttcttgtgag 2400

cgctggcctg ctagtggtca tcctgaagtt aggaaacctg ctgccctaca gtgatcccag 2460

cgtggtgttt gtcttcctgt ccgtgtttgc tgtggtgaca atcctgcagt gcttcctgat 2520

tagcacactc ttctccagag ccaacctggc agcagcctgt gggggcatca tctacttcac 2580

gctgtacctg ccctacgtcc tgtgtgtggc atggcaggac tacgtgggct tcacactcaa 2640

gatcttcgct agcctgctgt ctcctgtggc ttttgggttt ggctgtgagt actttgccct 2700

ttttgaggag cagggcattg gagtgcagtg ggacaacctg tttgagagtc ctgtggagga 2760

agatggcttc aatctcacca cttcggtctc catgatgctg tttgacacct tcctctatgg 2820

ggtgatgacc tggtacattg aggctgtctt tccaggccag tacggaattc ccaggccctg 2880

gtattttcct tgcaccaagt cctactggtt tggcgaggaa agtgatgaga agagccaccc 2940

tggttccaac cagaagagaa tatcagaaat ctgcatggag gaggaaccca cccacttgaa 3000

gctgggcgtg tccattcaga acctggtaaa agtctaccga gatgggatga aggtggctgt 3060

cgatggcctg gcactgaatt tttatgaggg ccagatcacc tccttcctgg gccacaatgg 3120

agcggggaag acgaccacca tgtcaatcct gaccgggttg ttccccccga cctcgggcac 3180

cgcctacatc ctgggaaaag acattcgctc tgagatgagc accatccggc agaacctggg 3240

ggtctgtccc cagcataacg tgctgtttga catgctgact gtcgaagaac acatctggtt 3300

ctatgcccgc ttgaaagggc tctctgagaa gcacgtgaag gcggagatgg agcagatggc 3360

cctggatgtt ggtttgccat caagcaagct gaaaagcaaa acaagccagc tgtcaggtgg 3420

aatgcagaga aagctatctg tggccttggc ctttgtcggg ggatctaagg ttgtcattct 3480

ggatgaaccc acagctggtg tggaccctta ctcccgcagg ggaatatggg agctgctgct 3540

gaaataccga caaggccgca ccattattct ctctacacac cacatggatg aagcggacgt 3600

cctgggggac aggattgcca tcatctccca tgggaagctg tgctgtgtgg gctcctccct 3660

gtttctgaag aaccagctgg gaacaggcta ctacctgacc ttggtcaaga aagatgtgga 3720

atcctccctc agttcctgca gaaacagtag tagcactgtg tcatacctga aaaaggagga 3780

cagtgtttct cagagcagtt ctgatgctgg cctgggcagc gaccatgaga gtgacacgct 3840

gaccatcgat gtctctgcta tctccaacct catcaggaag catgtgtctg aagcccggct 3900

ggtggaagac atagggcatg agctgaccta tgtgctgcca tatgaagctg ctaaggaggg 3960

agcctttgtg gaactctttc atgagattga tgaccggctc tcagacctgg gcatttctag 4020

ttatggcatc tcagagacga ccctggaaga aatattcctc aaggtggccg aagagagtgg 4080

ggtggatgct gagacctcag atggtacctt gccagcaaga cgaaacaggc gggccttcgg 4140

ggacaagcag agctgtcttc gcccgttcac tgaagatgat gctgctgatc caaatgattc 4200

tgacatagac ccagaatcca gagagacaga cttgctcagt gggatggatg gcaaagggtc 4260

ctaccaggtg aaaggctgga aacttacaca gcaacagttt gtggcccttt tgtggaagag 4320

actgctaatt gccagacgga gtcggaaagg attttttgct cagattgtct tgccagctgt 4380

gtttgtctgc attgcccttg tgttcagcct gatcgtgcca ccctttggca agtaccccag 4440

cctggaactt cagccctgga tgtacaacga acagtacaca tttgtcagca atgatgctcc 4500

tgaggacacg ggaaccctgg aactcttaaa cgccctcacc aaagaccctg gcttcgggac 4560

ccgctgtatg gaaggaaacc caatcccaga cacgccctgc caggcagggg aggaagagtg 4620

gaccactgcc ccagttcccc agaccatcat ggacctcttc cagaatggga actggacaat 4680

gcagaaccct tcacctgcat gccagtgtag cagcgacaaa atcaagaaga tgctgcctgt 4740

gtgtccccca ggggcagggg ggctgcctcc tccacaaaga aaacaaaaca ctgcagatat 4800

ccttcaggac ctgacaggaa gaaacatttc ggattatctg gtgaagacgt atgtgcagat 4860

catagccaaa agcttaaaga acaagatctg ggtgaatgag tttaggtatg gcggcttttc 4920

cctgggtgtc agtaatactc aagcacttcc tccgagtcaa gaagttaatg atgccatcaa 4980

acaaatgaag aaacacctaa agctggccaa ggacagttct gcagatcgat ttctcaacag 5040

cttgggaaga tttatgacag gactggacac caaaaataat gtcaaggtgt ggttcaataa 5100

caagggctgg catgcaatca gctctttcct gaatgtcatc aacaatgcca ttctccgggc 5160

caacctgcaa aagggagaga accctagcca ttatggaatt actgctttca atcatcccct 5220

gaatctcacc aagcagcagc tctcagaggt ggctctgatg accacatcag tggatgtcct 5280

tgtgtccatc tgtgtcatct ttgcaatgtc cttcgtccca gccagctttg tcgtattcct 5340

gatccaggag cgggtcagca aagcaaaaca cctgcagttc atcagtggag tgaagcctgt 5400

catctactgg ctctctaatt ttgtctggga tatgtgcaat tacgttgtcc ctgccacact 5460

ggtcattatc atcttcatct gcttccagca gaagtcctat gtgtcctcca ccaatctgcc 5520

tgtgctagcc cttctacttt tgctgtatgg gtggtcaatc acacctctca tgtacccagc 5580

ctcctttgtg ttcaagatcc ccagcacagc ctatgtggtg ctcaccagcg tgaacctctt 5640

cattggcatt aatggcagcg tggccacctt tgtgctggag ctgttcaccg acaataagct 5700

gaataatatc aatgatatcc tgaagtccgt gttcttgatc ttcccacatt tttgcctggg 5760

acgagggctc atcgacatgg tgaaaaacca ggcaatggct gatgccctgg aaaggtttgg 5820

ggagaatcgc tttgtgtcac cattatcttg ggacttggtg ggacgaaacc tcttcgccat 5880

ggccgtggaa ggggtggtgt tcttcctcat tactgttctg atccagtaca gattcttcat 5940

caggcccaga cctgtaaatg caaagctatc tcctctgaat gatgaagatg aagatgtgag 6000

gcgggaaaga cagagaattc ttgatggtgg aggccagaat gacatcttag aaatcaagga 6060

gttgacgaag atatatagaa ggaagcggaa gcctgctgtt gacaggattt gcgtgggcat 6120

tcctcctggt gagtgctttg ggctcctggg agttaatggg gctggaaaat catcaacttt 6180

caagatgtta acaggagata ccactgttac cagaggagat gctttcctta acaaaaatag 6240

tatcttatca aacatccatg aagtacatca gaacatgggc tactgccctc agtttgatgc 6300

catcacagag ctgttgactg ggagagaaca cgtggagttc tttgcccttt tgagaggagt 6360

cccagagaaa gaagttggca aggttggtga gtgggcgatt cggaaactgg gcctcgtgaa 6420

gtatggagaa aaatatgctg gtaactatag tggaggcaac aaacgcaagc tctctacagc 6480

catggctttg atcggcgggc ctcctgtggt gtttctggat gaacccacca caggcatgga 6540

tcccaaagcc cggcggttct tgtggaattg tgccctaagt gttgtcaagg aggggagatc 6600

agtagtgctt acatctcata gtatggaaga atgtgaagct ctttgcacta ggatggcaat 6660

catggtcaat ggaaggttca ggtgccttgg cagtgtccag catctaaaaa ataggtttgg 6720

agatggttat acaatagttg tacgaatagc agggtccaac ccggacctga agcctgtcca 6780

ggatttcttt ggacttgcat ttcctggaag tgttctaaaa gagaaacacc ggaacatgct 6840

acaataccag cttccatctt cattatcttc tctggccagg atattcagca tcctctccca 6900

gagcaaaaag cgactccaca tagaagacta ctctgtttct cagacaacac ttgaccaagt 6960

atttgtgaac tttgccaagg accaaagtga tgatgaccac ttaaaagacc tctcattaca 7020

caaaaaccag acagtagtgg acgttgcagt tctcacatct tttctacagg atgagaaagt 7080

gaaagaaagc tatgtatgaa gaatcctgtt catacggggt ggctgaaagt aaagaggaac 7140

tagactttcc tttgcaccat gtgaagtgtt gtggagaaaa gagccagaag ttgatgtggg 7200

aagaagtaaa ctggatactg tactgatact attcaatgca atgcaattca atgcaatgaa 7260

aacaaaattc cattacaggg gcagtgcctt tgtagcctat gtcttgtatg gctctcaagt 7320

gaaagacttg aatttagttt tttacctata cctatgtgaa actctattat ggaacccaat 7380

ggacatatgg gtttgaactc acactttttt tttttttttt gttcctgtgt attctcattg 7440

gggttgcaac aataattcat caagtaatca tggccagcga ttattgatca aaatcaaaag 7500

gtaatgcaca tcctcattca ctaagccatg ccatgcccag gagactggtt tcccggtgac 7560

acatccattg ctggcaatga gtgtgccaga gttattagtg ccaagttttt cagaaagttt 7620

gaagcaccat ggtgtgtcat gctcactttt gtgaaagctg ctctgctcag agtctatcaa 7680

cattgaatat cagttgacag aatggtgcca tgcgtggcta acatcctgct ttgattccct 7740

ctgataagct gttctggtgg cagtaacatg caacaaaaat gtgggtgtct ccaggcacgg 7800

gaaacttggt tccattgtta tattgtccta tgcttcgagc catgggtcta cagggtcatc 7860

cttatgagac tcttaaatat acttagatcc tggtaagagg caaagaatca acagccaaac 7920

tgctggggct gcaagctgct gaagccaggg catgggatta aagagattgt gcgttcaaac 7980

ctagggaagc ctgtgcccat ttgtcctgac tgtctgctaa catggtacac tgcatctcaa 8040

gatgtttatc tgacacaagt gtattatttc tggctttttg aattaatcta gaaaatgaaa 8100

agatggagtt gtattttgac aaaaatgttt gtacttttta atgttatttg gaattttaag 8160

ttctatcagt gacttctgaa tccttagaat ggcctctttg tagaaccctg tggtatagag 8220

gagtatggcc actgccccac tatttttatt ttcttatgta agtttgcata tcagtcatga 8280

ctagtgccta gaaagcaatg tgatggtcag gatctcatga cattatattt gagtttcttt 8340

cagatcattt aggatactct taatctcact tcatcaatca aatatttttt gagtgtatgc 8400

tgtagctgaa agagtatgta cgtacgtata agactagaga gatattaagt ctcagtacac 8460

ttcctgtgcc atgttattca gctcactggt ttacaaatat aggttgtctt gtggttgtag 8520

gagcccactg taacaatact gggcagcctt tttttttttt tttttaattg caacaatgca 8580

aaagccaaga aagtataagg gtcacaagtc taaacaatga attcttcaac agggaaaaca 8640

gctagcttga aaacttgctg aaaaacacaa cttgtgttta tggcatttag taccttcaaa 8700

taattggctt tgcagatatt ggatacccca ttaaatctga cagtctcaaa tttttcatct 8760

cttcaatcac tagtcaagaa aaatataaaa acaacaaata cttccatatg gagcattttt 8820

cagagttttc taacccagtc ttatttttct agtcagtaaa catttgtaaa aatactgttt 8880

cactaatact tactgttaac tgtcttgaga gaaaagaaaa atatgagaga actattgttt 8940

ggggaagttc aagtgatctt tcaatatcat tactaacttc ttccactttt tccagaattt 9000

gaatattaac gctaaaggtg taagacttca gatttcaaat taatctttct atatttttta 9060

aatttacaga atattatata acccactgct gaaaaagaaa aaaatgattg ttttagaagt 9120

taaagtcaat attgatttta aatataagta atgaaggcat atttccaata actagtgata 9180

tggcatcgtt gcattttaca gtatcttcaa aaatacagaa tttatagaat aatttctcct 9240

catttaatat ttttcaaaat caaagttatg gtttcctcat tttactaaaa tcgtattcta 9300

attcttcatt atagtaaatc tatgagcaac tccttacttc ggttcctctg atttcaaggc 9360

catattttaa aaaatcaaaa ggcactgtga actattttga agaaaacaca acattttaat 9420

acagattgaa aggacctctt ctgaagctag aaacaatcta tagttataca tcttcattaa 9480

tactgtgtta ccttttaaaa tagtaatttt ttacattttc ctgtgtaaac ctaattgtgg 9540

tagaaatttt taccaactct atactcaatc aagcaaaatt tctgtatatt ccctgtggaa 9600

tgtacctatg tgagtttcag aaattctcaa aatacgtgtt caaaaatttc tgcttttgca 9660

tctttgggac acctcagaaa acttattaac aactgtgaat atgagaaata cagaagaaaa 9720

taataagccc tctatacata aatgcccagc acaattcatt gttaaaaaac aaccaaacct 9780

cacactactg tatttcatta tctgtactga aagcaaatgc tttgtgacta ttaaatgttg 9840

cacatcattc attcactgta tagtaatcat tgactaaagc catttgtctg tgttttcttc 9900

ttgtggttgt atatatcagg taaaatattt tccaaagagc catgtgtcat gtaatactga 9960

accactttga tattgagaca ttaatttgta cccttgttat tatctactag taataatgta 10020

atactgtaga aatattgctc taattctttt caaaattgtt gcatccccct tagaatgttt 10080

ctatttccat aaggatttag gtatgctatt atcccttctt ataccctaag atgaagctgt 10140

ttttgtgctc tttgttcatc attggccctc attccaagca ctttacgctg tctgtaatgg 10200

gatctatttt tgcactggaa tatctgagaa ttgcaaaact agacaaaagt ttcacaacag 10260

atttctaagt taaatcattt tcattaaaag gaaaaaagaa aaaaaatttt gtatgtcaat 10320

aactttatat gaagtattaa aatgcatatt tctatgttgt aatataatga gtcacaaaat 10380

aaagctgtga cagttctgtt ggtctacaga aa 10412

<210> 2

<211> 1354

<212> DNA

＜213〉homo sapiens

<300>

<308> NM_000229.1

<309> 2010-08-02

<313> (1)..(1354)

<400> 2

ccagggctgg aatggggccg cccggctccc catggcagtg ggtgacgctg ctgctggggc 60

tgctgctccc tcctgccgcc cccttctggc tcctcaatgt gctcttcccc ccgcacacca 120

cgcccaaggc tgagctcagt aaccacacac ggcccgtcat cctcgtgccc ggctgcctgg 180

ggaatcagct agaagccaag ctggacaaac cagatgtggt gaactggatg tgctaccgca 240

agacagagga cttcttcacc atctggctgg atctcaacat gttcctaccc cttggggtag 300

actgctggat cgataacacc agggttgtct acaaccggag ctctgggctc gtgtccaacg 360

cccctggtgt ccagatccgc gtccctggct ttggcaagac ctactctgtg gagtacctgg 420

acagcagcaa gctggcaggg tacctgcaca cactggtgca gaacctggtc aacaatggct 480

acgtgcggga cgagactgtg cgcgccgccc cctatgactg gcggctggag cccggccagc 540

aggaggagta ctaccgcaag ctcgcagggc tggtggagga gatgcacgct gcctatggga 600

agcctgtctt cctcattggc cacagcctcg gctgtctaca cttgctctat ttcctgctgc 660

gccagcccca ggcctggaag gaccgcttta ttgatggctt catctctctt ggggctccct 720

ggggtggctc catcaagccc atgctggtct tggcctcagg tgacaaccag ggcatcccca 780

tcatgtccag catcaagctg aaagaggagc agcgcataac caccacctcc ccctggatgt 840

ttccctctcg catggcgtgg cctgaggacc acgtgttcat ttccacaccc agcttcaact 900

acacaggccg tgacttccaa cgcttctttg cagacctgca ctttgaggaa ggctggtaca 960

tgtggctgca gtcacgtgac ctcctggcag gactcccagc acctggtgtg gaagtatact 1020

gtctttacgg cgtgggcctg cccacgcccc gcacctacat ctacgaccac ggcttcccct 1080

acacggaccc tgtgggtgtg ctctatgagg atggtgatga cacggtggcg acccgcagca 1140

ccgagctctg tggcctgtgg cagggccgcc agccacagcc tgtgcacctg ctgcccctgc 1200

acgggataca gcatctcaac atggtcttca gcaacctgac cctggagcac atcaatgcca 1260

tcctgctggg tgcctaccgc cagggtcccc ctgcatcccc gactgccagc ccagagcccc 1320

cgcctcctga ataaagacct tcctttgcta ccgt 1354

<210> 3

<211> 14905

<212> DNA

＜213〉homo sapiens

<300>

<308> NM_002332.2

<309> 2010-08-04

<313> (1)..(14905)

<400> 3

cagcggtgcg agctccaggc ccatgcactg aggaggcgga aacaagggga gcccccagag 60

ctccatcaag ccccctccaa aggctcccct acccggtcca cgccccccac cccccctccc 120

cgcctcctcc caattgtgca tttttgcagc cggaggcggc tccgagatgg ggctgtgagc 180

ttcgcccggg gagggggaaa gagcagcgag gagtgaagcg ggggggtggg gtgaagggtt 240

tggatttcgg ggcagggggc gcacccccgt cagcaggccc tccccaaggg gctcggaact 300

ctacctcttc acccacgccc ctggtgcgct ttgccgaagg aaagaataag aacagagaag 360

gaggaggggg aaaggaggaa aagggggacc ccccaactgg ggggggtgaa ggagagaagt 420

agcaggacca gaggggaagg ggctgctgct tgcatcagcc cacaccatgc tgaccccgcc 480

gttgctcctg ctgctgcccc tgctctcagc tctggtcgcg gcggctatcg acgcccctaa 540

gacttgcagc cccaagcagt ttgcctgcag agatcaaata acctgtatct caaagggctg 600

gcggtgcgac ggtgagaggg actgcccaga cggatctgac gaggcccctg agatttgtcc 660

acagagtaag gcccagcgat gccagccaaa cgagcataac tgcctgggta ctgagctgtg 720

tgttcccatg tcccgcctct gcaatggggt ccaggactgc atggacggct cagatgaggg 780

gccccactgc cgagagctcc aaggcaactg ctctcgcctg ggctgccagc accattgtgt 840

ccccacactc gatgggccca cctgctactg caacagcagc tttcagcttc aggcagatgg 900

caagacctgc aaagattttg atgagtgctc agtgtacggc acctgcagcc agctatgcac 960

caacacagac ggctccttca tatgtggctg tgttgaagga tacctcctgc agccggataa 1020

ccgctcctgc aaggccaaga acgagccagt agaccggccc cctgtgctgt tgatagccaa 1080

ctcccagaac atcttggcca cgtacctgag tggggcccag gtgtctacca tcacacctac 1140

gagcacgcgg cagaccacag ccatggactt cagctatgcc aacgagaccg tatgctgggt 1200

gcatgttggg gacagtgctg ctcagacgca gctcaagtgt gcccgcatgc ctggcctaaa 1260

gggcttcgtg gatgagcaca ccatcaacat ctccctcagt ctgcaccacg tggaacagat 1320

ggccatcgac tggctgacag gcaacttcta ctttgtggat gacatcgatg ataggatctt 1380

tgtctgcaac agaaatgggg acacatgtgt cacattgcta gacctggaac tctacaaccc 1440

caagggcatt gccctggacc ctgccatggg gaaggtgttt ttcactgact atgggcagat 1500

cccaaaggtg gaacgctgtg acatggatgg gcagaaccgc accaagctcg tcgacagcaa 1560

gattgtgttt cctcatggca tcacgctgga cctggtcagc cgccttgtct actgggcaga 1620

tgcctatctg gactatattg aagtggtgga ctatgagggc aagggccgcc agaccatcat 1680

ccagggcatc ctgattgagc acctgtacgg cctgactgtg tttgagaatt atctctatgc 1740

caccaactcg gacaatgcca atgcccagca gaagacgagt gtgatccgtg tgaaccgctt 1800

taacagcacc gagtaccagg ttgtcacccg ggtggacaag ggtggtgccc tccacatcta 1860

ccaccagagg cgtcagcccc gagtgaggag ccatgcctgt gaaaacgacc agtatgggaa 1920

gccgggtggc tgctctgaca tctgcctgct ggccaacagc cacaaggcgc ggacctgccg 1980

ctgccgttcc ggcttcagcc tgggcagtga cgggaagtca tgcaagaagc cggagcatga 2040

gctgttcctc gtgtatggca agggccggcc aggcatcatc cggggcatgg atatgggggc 2100

caaggtcccg gatgagcaca tgatccccat tgaaaacctc atgaaccccc gagccctgga 2160

cttccacgct gagaccggct tcatctactt tgccgacacc accagctacc tcattggccg 2220

ccagaagatt gatggcactg agcgggagac catcctgaag gacggcatcc acaatgtgga 2280

gggtgtggcc gtggactgga tgggagacaa tctgtactgg acggacgatg ggcccaaaaa 2340

gacaatcagc gtggccaggc tggagaaagc tgctcagacc cgcaagactt taatcgaggg 2400

caaaatgaca caccccaggg ctattgtggt ggatccactc aatgggtgga tgtactggac 2460

agactgggag gaggacccca aggacagtcg gcgtgggcgg ctggagaggg cgtggatgga 2520

tggctcacac cgagacatct ttgtcacctc caagacagtg ctttggccca atgggctaag 2580

cctggacatc ccggctgggc gcctctactg ggtggatgcc ttctacgacc gcatcgagac 2640

gatactgctc aatggcacag accggaagat tgtgtatgaa ggtcctgagc tgaaccacgc 2700

ctttggcctg tgtcaccatg gcaactacct cttctggact gagtatcgga gtggcagtgt 2760

ctaccgcttg gaacggggtg taggaggcgc accccccact gtgacccttc tgcgcagtga 2820

gcggcccccc atctttgaga tccgaatgta tgatgcccag cagcagcaag ttggcaccaa 2880

caaatgccgg gtgaacaatg gcggctgcag cagcctgtgc ttggccaccc ctgggagccg 2940

ccagtgcgcc tgtgctgagg accaggtgtt ggacgcagac ggcgtcactt gcttggcgaa 3000

cccatcctac gtgcctccac cccagtgcca gccaggcgag tttgcctgtg ccaacagccg 3060

ctgcatccag gagcgctgga agtgtgacgg agacaacgat tgcctggaca acagtgatga 3120

ggccccagcc ctctgccatc agcacacctg cccctcggac cgattcaagt gcgagaacaa 3180

ccggtgcatc cccaaccgct ggctctgcga cggggacaat gactgtggga acagtgaaga 3240

tgagtccaat gccacttgtt cagcccgcac ctgccccccc aaccagttct cctgtgccag 3300

tggccgctgc atccccatct cctggacgtg tgatctggat gacgactgtg gggaccgctc 3360

tgatgagtct gcttcgtgtg cctatcccac ctgcttcccc ctgactcagt ttacctgcaa 3420

caatggcaga tgtatcaaca tcaactggag atgcgacaat gacaatgact gtggggacaa 3480

cagtgacgaa gccggctgca gccactcctg ttctagcacc cagttcaagt gcaacagcgg 3540

gcgttgcatc cccgagcact ggacctgcga tggggacaat gactgcggag actacagtga 3600

tgagacacac gccaactgca ccaaccaggc cacgaggccc cctggtggct gccacactga 3660

tgagttccag tgccggctgg atggactatg catccccctg cggtggcgct gcgatgggga 3720

cactgactgc atggactcca gcgatgagaa gagctgtgag ggagtgaccc acgtctgcga 3780

tcccagtgtc aagtttggct gcaaggactc agctcggtgc atcagcaaag cgtgggtgtg 3840

tgatggcgac aatgactgtg aggataactc ggacgaggag aactgcgagt ccctggcctg 3900

caggccaccc tcgcaccctt gtgccaacaa cacctcagtc tgcctgcccc ctgacaagct 3960

gtgtgatggc aacgacgact gtggcgacgg ctcagatgag ggcgagctct gcgaccagtg 4020

ctctctgaat aacggtggct gcagccacaa ctgctcagtg gcacctggcg aaggcattgt 4080

gtgttcctgc cctctgggca tggagctggg gcccgacaac cacacctgcc agatccagag 4140

ctactgtgcc aagcatctca aatgcagcca aaagtgcgac cagaacaagt tcagcgtgaa 4200

gtgctcctgc tacgagggct gggtcctgga acctgacggc gagagctgcc gcagcctgga 4260

ccccttcaag ccgttcatca ttttctccaa ccgccatgaa atccggcgca tcgatcttca 4320

caaaggagac tacagcgtcc tggtgcccgg cctgcgcaac accatcgccc tggacttcca 4380

cctcagccag agcgccctct actggaccga cgtggtggag gacaagatct accgcgggaa 4440

gctgctggac aacggagccc tgactagttt cgaggtggtg attcagtatg gcctggccac 4500

acccgagggc ctggctgtag actggattgc aggcaacatc tactgggtgg agagtaacct 4560

ggatcagatc gaggtggcca agctggatgg gaccctccgg accaccctgc tggccggtga 4620

cattgagcac ccaagggcaa tcgcactgga tccccgggat gggatcctgt tttggacaga 4680

ctgggatgcc agcctgcccc gcattgaggc agcctccatg agtggggctg ggcgccgcac 4740

cgtgcaccgg gagaccggct ctgggggctg gcccaacggg ctcaccgtgg actacctgga 4800

gaagcgcatc ctttggattg acgccaggtc agatgccatt tactcagccc gttacgacgg 4860

ctctggccac atggaggtgc ttcggggaca cgagttcctg tcgcacccgt ttgcagtgac 4920

gctgtacggg ggggaggtct actggactga ctggcgaaca aacacactgg ctaaggccaa 4980

caagtggacc ggccacaatg tcaccgtggt acagaggacc aacacccagc cctttgacct 5040

gcaggtgtac cacccctccc gccagcccat ggctcccaat ccctgtgagg ccaatggggg 5100

ccagggcccc tgctcccacc tgtgtctcat caactacaac cggaccgtgt cctgcgcctg 5160

cccccacctc atgaagctcc acaaggacaa caccacctgc tatgagttta agaagttcct 5220

gctgtacgca cgtcagatgg agatccgagg tgtggacctg gatgctccct actacaacta 5280

catcatctcc ttcacggtgc ccgacatcga caacgtcaca gtgctagact acgatgcccg 5340

cgagcagcgt gtgtactggt ctgacgtgcg gacacaggcc atcaagcggg ccttcatcaa 5400

cggcacaggc gtggagacag tcgtctctgc agacttgcca aatgcccacg ggctggctgt 5460

ggactgggtc tcccgaaacc tgttctggac aagctatgac accaataaga agcagatcaa 5520

tgtggcccgg ctggatggct ccttcaagaa cgcagtggtg cagggcctgg agcagcccca 5580

tggccttgtc gtccaccctc tgcgtgggaa gctctactgg accgatggtg acaacatcag 5640

catggccaac atggatggca gcaatcgcac cctgctcttc agtggccaga agggccccgt 5700

gggcctggct attgacttcc ctgaaagcaa actctactgg atcagctccg ggaaccatac 5760

catcaaccgc tgcaacctgg atgggagtgg gctggaggtc atcgatgcca tgcggagcca 5820

gctgggcaag gccaccgccc tggccatcat gggggacaag ctgtggtggg ctgatcaggt 5880

gtcggaaaag atgggcacat gcagcaaggc tgacggctcg ggctccgtgg tccttcggaa 5940

cagcaccacc ctggtgatgc acatgaaggt ctatgacgag agcatccagc tggaccataa 6000

gggcaccaac ccctgcagtg tcaacaacgg tgactgctcc cagctctgcc tgcccacgtc 6060

agagacgacc cgctcctgca tgtgcacagc cggctatagc ctccggagtg gccagcaggc 6120

ctgcgagggc gtaggttcct ttctcctgta ctctgtgcat gagggaatca ggggaattcc 6180

cctggatccc aatgacaagt cagatgccct ggtcccagtg tccgggacct cgctggctgt 6240

cggcatcgac ttccacgctg aaaatgacac catctactgg gtggacatgg gcctgagcac 6300

gatcagccgg gccaagcggg accagacgtg gcgtgaagac gtggtgacca atggcattgg 6360

ccgtgtggag ggcattgcag tggactggat cgcaggcaac atctactgga cagaccaggg 6420

ctttgatgtc atcgaggtcg cccggctcaa tggctccttc cgctacgtgg tgatctccca 6480

gggtctagac aagccccggg ccatcaccgt ccacccggag aaagggtact tgttctggac 6540

tgagtggggt cagtatccgc gtattgagcg gtctcggcta gatggcacgg agcgtgtggt 6600

gctggtcaac gtcagcatca gctggcccaa cggcatctca gtggactacc aggatgggaa 6660

gctgtactgg tgcgatgcac ggacagacaa gattgaacgg atcgacctgg agacaggtga 6720

gaaccgcgag gtggttctgt ccagcaacaa catggacatg ttttcagtgt ctgtgtttga 6780

ggatttcatc tactggagtg acaggactca tgccaacggc tctatcaagc gcgggagcaa 6840

agacaatgcc acagactccg tgcccctgcg aaccggcatc ggcgtccagc ttaaagacat 6900

caaagtcttc aaccgggacc ggcagaaagg caccaacgtg tgcgcggtgg ccaatggcgg 6960

gtgccagcag ctgtgcctgt accggggccg tgggcagcgg gcctgcgcct gtgcccacgg 7020

gatgctggct gaagacggag catcgtgccg cgagtatgcc ggctacctgc tctactcaga 7080

gcgcaccatt ctcaagagta tccacctgtc ggatgagcgc aacctcaatg cgcccgtgca 7140

gcccttcgag gaccctgagc acatgaagaa cgtcatcgcc ctggcctttg actaccgggc 7200

aggcacctct ccgggcaccc ccaatcgcat cttcttcagc gacatccact ttgggaacat 7260

ccaacagatc aacgacgatg gctccaggag gatcaccatt gtggaaaacg tgggctccgt 7320

ggaaggcctg gcctatcacc gtggctggga cactctctat tggacaagct acacgacatc 7380

caccatcacg cgccacacag tggaccagac ccgcccaggg gccttcgagc gtgagaccgt 7440

catcactatg tctggagatg accacccacg ggccttcgtt ttggacgagt gccagaacct 7500

catgttctgg accaactgga atgagcagca tcccagcatc atgcgggcgg cgctctcggg 7560

agccaatgtc ctgaccctta tcgagaagga catccgtacc cccaatggcc tggccatcga 7620

ccaccgtgcc gagaagctct acttctctga cgccaccctg gacaagatcg agcggtgcga 7680

gtatgacggc tcccaccgct atgtgatcct aaagtcagag cctgtccacc ccttcgggct 7740

ggccgtgtat ggggagcaca ttttctggac tgactgggtg cggcgggcag tgcagcgggc 7800

caacaagcac gtgggcagca acatgaagct gctgcgcgtg gacatccccc agcagcccat 7860

gggcatcatc gccgtggcca acgacaccaa cagctgtgaa ctctctccat gccgaatcaa 7920

caacggtggc tgccaggacc tgtgtctgct cactcaccag ggccatgtca actgctcatg 7980

ccgagggggc cgaatcctcc aggatgacct cacctgccga gcggtgaatt cctcttgccg 8040

agcacaagat gagtttgagt gtgccaatgg cgagtgcatc aacttcagcc tgacctgcga 8100

cggcgtcccc cactgcaagg acaagtccga tgagaagcca tcctactgca actcccgccg 8160

ctgcaagaag actttccggc agtgcagcaa tgggcgctgt gtgtccaaca tgctgtggtg 8220

caacggggcc gacgactgtg gggatggctc tgacgagatc ccttgcaaca agacagcctg 8280

tggtgtgggc gagttccgct gccgggacgg gacctgcatc gggaactcca gccgctgcaa 8340

ccagtttgtg gattgtgagg acgcctcaga tgagatgaac tgcagtgcca ccgactgcag 8400

cagctacttc cgcctgggcg tgaagggcgt gctcttccag ccctgcgagc ggacctcact 8460

ctgctacgca cccagctggg tgtgtgatgg cgccaatgac tgtggggact acagtgatga 8520

gcgcgactgc ccaggtgtga aacgccccag atgccctctg aattacttcg cctgccctag 8580

tgggcgctgc atccccatga gctggacgtg tgacaaagag gatgactgtg aacatggcga 8640

ggacgagacc cactgcaaca agttctgctc agaggcccag tttgagtgcc agaaccatcg 8700

ctgcatctcc aagcagtggc tgtgtgacgg cagcgatgac tgtggggatg gctcagacga 8760

ggctgctcac tgtgaaggca agacgtgcgg cccctcctcc ttctcctgcc ctggcaccca 8820

cgtgtgcgtc cccgagcgct ggctctgtga cggtgacaaa gactgtgctg atggtgcaga 8880

cgagagcatc gcagctggtt gcttgtacaa cagcacttgt gacgaccgtg agttcatgtg 8940

ccagaaccgc cagtgcatcc ccaagcactt cgtgtgtgac cacgaccgtg actgtgcaga 9000

tggctctgat gagtcccccg agtgtgagta cccgacctgc ggccccagtg agttccgctg 9060

tgccaatggg cgctgtctga gctcccgcca gtgggagtgt gatggcgaga atgactgcca 9120

cgaccagagt gacgaggctc ccaagaaccc acactgcacc agccaagagc acaagtgcaa 9180

tgcctcgtca cagttcctgt gcagcagtgg gcgctgtgtg gctgaggcac tgctctgcaa 9240

cggccaggat gactgtggcg acagctcgga cgagcgtggc tgccacatca atgagtgtct 9300

cagccgcaag ctcagtggct gcagccagga ctgtgaggac ctcaagatcg gcttcaagtg 9360

ccgctgtcgc cctggcttcc ggctgaagga cgacggccgg acgtgtgctg atgtggacga 9420

gtgcagcacc accttcccct gcagccagcg ctgcatcaac actcatggca gctataagtg 9480

tctgtgtgtg gagggctatg caccccgcgg cggcgacccc cacagctgca aggctgtgac 9540

tgacgaggaa ccgtttctga tcttcgccaa ccggtactac ctgcgcaagc tcaacctgga 9600

cgggtccaac tacacgttac ttaagcaggg cctgaacaac gccgttgcct tggattttga 9660

ctaccgagag cagatgatct actggacaga tgtgaccacc cagggcagca tgatccgaag 9720

gatgcacctt aacgggagca atgtgcaggt cctacaccgt acaggcctca gcaaccccga 9780

tgggctggct gtggactggg tgggtggcaa cctgtactgg tgcgacaaag gccgggacac 9840

catcgaggtg tccaagctca atggggccta tcggacggtg ctggtcagct ctggcctccg 9900

tgagcccagg gctctggtgg tggatgtgca gaatgggtac ctgtactgga cagactgggg 9960

tgaccattca ctgatcggcc gcatcggcat ggatgggtcc agccgcagcg tcatcgtgga 10020

caccaagatc acatggccca atggcctgac gctggactat gtcactgagc gcatctactg 10080

ggccgacgcc cgcgaggact acattgaatt tgccagcctg gatggctcca atcgccacgt 10140

tgtgctgagc caggacatcc cgcacatctt tgcactgacc ctgtttgagg actacgtcta 10200

ctggaccgac tgggaaacaa agtccattaa ccgagcccac aagaccacgg gcaccaacaa 10260

aacgctcctc atcagcacgc tgcaccggcc catggacctg catgtcttcc atgccctgcg 10320

ccagccagac gtgcccaatc acccctgcaa ggtcaacaat ggtggctgca gcaacctgtg 10380

cctgctgtcc cccgggggag ggcacaaatg tgcctgcccc accaacttct acctgggcag 10440

cgatgggcgc acctgtgtgt ccaactgcac ggctagccag tttgtatgca agaacgacaa 10500

gtgcatcccc ttctggtgga agtgtgacac cgaggacgac tgcggggacc actcagacga 10560

gcccccggac tgccctgagt tcaagtgccg gcccggacag ttccagtgct ccacaggtat 10620

ctgcacaaac cctgccttca tctgcgatgg cgacaatgac tgccaggaca acagtgacga 10680

ggccaactgt gacatccacg tctgcttgcc cagtcagttc aaatgcacca acaccaaccg 10740

ctgtattccc ggcatcttcc gctgcaatgg gcaggacaac tgcggagatg gggaggatga 10800

gagggactgc cccgaggtga cctgcgcccc caaccagttc cagtgctcca ttaccaaacg 10860

gtgcatcccc cgggtctggg tctgcgaccg ggacaatgac tgtgtggatg gcagtgatga 10920

gcccgccaac tgcacccaga tgacctgtgg tgtggacgag ttccgctgca aggattcggg 10980

ccgctgcatc ccagcgcgtt ggaagtgtga cggagaggat gactgtgggg atggctcgga 11040

tgagcccaag gaagagtgtg atgaacgcac ctgtgagcca taccagttcc gctgcaagaa 11100

caaccgctgc gtgcccggcc gctggcagtg cgactacgac aacgattgcg gtgacaactc 11160

cgatgaagag agctgcaccc ctcggccctg ctccgagagt gagttctcct gtgccaacgg 11220

ccgctgcatc gcggggcgct ggaaatgcga tggagaccac gactgcgcgg acggctcgga 11280

cgagaaagac tgcacccccc gctgtgacat ggaccagttc cagtgcaaga gcggccactg 11340

catccccctg cgctggcgct gtgacgcaga cgccgactgc atggacggca gcgacgagga 11400

ggcctgcggc actggcgtgc ggacctgccc cctggacgag ttccagtgca acaacacctt 11460

gtgcaagccg ctggcctgga agtgcgatgg cgaggatgac tgtggggaca actcagatga 11520

gaaccccgag gagtgtgccc ggttcgtgtg ccctcccaac cggcccttcc gttgcaagaa 11580

tgaccgcgtc tgtctgtgga tcgggcgcca atgcgatggc acggacaact gtggggatgg 11640

gactgatgaa gaggactgtg agccccccac agcccacacc acccactgca aagacaagaa 11700

ggagtttctg tgccggaacc agcgctgcct ctcctcctcc ctgcgctgca acatgttcga 11760

tgactgcggg gacggctctg acgaggagga ctgcagcatc gaccccaagc tgaccagctg 11820

cgccaccaat gccagcatct gtggggacga ggcacgctgc gtgcgcaccg agaaagcggc 11880

ctactgtgcc tgccgctcgg gcttccacac cgtgcccggc cagcccggat gccaagacat 11940

caacgagtgc ctgcgcttcg gcacctgctc ccagctctgc aacaacacca agggcggcca 12000

cctctgcagc tgcgctcgga acttcatgaa gacgcacaac acctgcaagg ccgaaggctc 12060

tgagtaccag gtcctgtaca tcgctgatga caatgagatc cgcagcctgt tccccggcca 12120

cccccattcg gcttacgagc aggcattcca gggtgacgag agtgtccgca ttgatgctat 12180

ggatgtccat gtcaaggctg gccgtgtcta ttggaccaac tggcacacgg gcaccatctc 12240

ctaccgcagc ctgccacctg ctgcgcctcc taccacttcc aaccgccacc ggcgacagat 12300

tgaccggggt gtcacccacc tcaacatttc agggctgaag atgcccagag gcatcgccat 12360

cgactgggtg gccggaaacg tgtactggac cgactcgggc cgagatgtga ttgaggtggc 12420

gcagatgaag ggcgagaacc gcaagacgct catctcgggc atgattgacg agccccacgc 12480

cattgtggtg gacccactga gggggaccat gtactggtca gactggggca accaccccaa 12540

gattgagacg gcagcgatgg atgggacgct tcgggagaca ctggtgcagg acaacattca 12600

gtggcccaca ggcctggccg tggattatca caatgagcgg ctgtactggg cagacgccaa 12660

gctttcagtc atcggcagca tccggctcaa tggcacggac cccattgtgg ctgctgacag 12720

caaacgaggc ctaagtcacc ccttcagcat cgacgtcttt gaggattaca tctatggtgt 12780

cacctacatc aataatcgtg tcttcaagat ccataagttt ggccacagcc ccttggtcaa 12840

cctgacaggg ggcctgagcc acgcctctga cgtggtcctt taccatcagc acaagcagcc 12900

cgaagtgacc aacccatgtg accgcaagaa atgcgagtgg ctctgcctgc tgagccccag 12960

tgggcctgtc tgcacctgtc ccaatgggaa gcggctggac aacggcacat gcgtgcctgt 13020

gccctctcca acgccccccc cagatgctcc ccggcctgga acctgtaacc tgcagtgctt 13080

caacggtggc agctgtttcc tcaatgcacg gaggcagccc aagtgccgct gccaaccccg 13140

ctacacgggt gacaagtgtg aactggacca gtgctgggag cactgtcgca atgggggcac 13200

ctgtgctgcc tccccctctg gcatgcccac gtgccggtgc cccacgggct tcacgggccc 13260

caaatgcacc cagcaggtgt gtgcgggcta ctgtgccaac aacagcacct gcactgtcaa 13320

ccagggcaac cagccccagt gccgatgcct acccggcttc ctgggcgacc gctgccagta 13380

ccggcagtgc tctggctact gtgagaactt tggcacatgc cagatggctg ctgatggctc 13440

ccgacaatgc cgctgcactg cctactttga gggatcgagg tgtgaggtga acaagtgcag 13500

ccgctgtctc gaaggggcct gtgtggtcaa caagcagagt ggggatgtca cctgcaactg 13560

cacggatggc cgggtggccc ccagctgtct gacctgcgtc ggccactgca gcaatggcgg 13620

ctcctgtacc atgaacagca aaatgatgcc tgagtgccag tgcccacccc acatgacagg 13680

gccccggtgt gaggagcacg tcttcagcca gcagcagcca ggacatatag cctccatcct 13740

aatccctctg ctgttgctgc tgctgctggt tctggtggcc ggagtggtat tctggtataa 13800

gcggcgagtc caaggggcta agggcttcca gcaccaacgg atgaccaacg gggccatgaa 13860

cgtggagatt ggaaacccca cctacaagat gtacgaaggc ggagagcctg atgatgtggg 13920

aggcctactg gacgctgact ttgccctgga ccctgacaag cccaccaact tcaccaaccc 13980

cgtgtatgcc acactctaca tggggggcca tggcagtcgc cactccctgg ccagcacgga 14040

cgagaagcga gaactcctgg gccggggccc tgaggacgag ataggggacc ccttggcata 14100

gggccctgcc ccgtcggact gcccccagaa agcctcctgc cccctgccgg tgaagtcctt 14160

cagtgagccc ctccccagcc agcccttccc tggccccgcc ggatgtataa atgtaaaaat 14220

gaaggaatta cattttatat gtgagcgagc aagccggcaa gcgagcacag tattatttct 14280

ccatcccctc cctgcctgct ccttggcacc cccatgctgc cttcagggag acaggcaggg 14340

agggcttggg gctgcacctc ctaccctccc accagaacgc accccactgg gagagctggt 14400

ggtgcagcct tcccctccct gtataagaca ctttgccaag gctctcccct ctcgccccat 14460

ccctgcttgc ccgctcccac agcttcctga gggctaattc tgggaaggga gagttctttg 14520

ctgcccctgt ctggaagacg tggctctggg tgaggtaggc gggaaaggat ggagtgtttt 14580

agttcttggg ggaggccacc ccaaacccca gccccaactc caggggcacc tatgagatgg 14640

ccatgctcaa cccccctccc agacaggccc tccctgtctc cagggccccc accgaggttc 14700

ccagggctgg agacttcctc tggtaaacat tcctccagcc tcccctcccc tggggacgcc 14760

aaggaggtgg gccacaccca ggaagggaaa gcgggcagcc ccgttttggg gacgtgaacg 14820

ttttaataat ttttgctgaa ttcctttaca actaaataac acagatattg ttataaataa 14880

aattgtaaaa aaaaaaaaaa aaaaa 14905

<210> 4

<211> 14907

<212> DNA

＜213〉house mouse

<300>

<308> NM_008512.2

<309> 2010-05-16

<313> (1)..(14907)

<400> 4

agtcagggga gcagcggtgc gagctccagg ccagtgcact gaggaggcgg aaacggggga 60

gcccctagtg ctccatcagg cccctaccaa ggcaccccca tcgggtccac gccccccacc 120

ccccaccccg cctcctccca attgtgcatt tttgcagcca gaggcggctc cgagatgggg 180

ctgtgagctt cgccctggga gggggagagg agcgaggagt aaagcagggg tgaagggttc 240

gaatttgggg gcagggggcg cacccgcgtc agcaggccct tcccaggggg ctcggaactg 300

taccatttca cctatgcccc tggttcgctt tgcttaagga aaggataaga atagaagagt 360

cggggagagg aagataaagg gggacccccc aattgggggg ggcgaggaca agaagtaaca 420

ggaccagagg gtgggggctg ctgtttgcat cggcccacac catgctgacc ccgccgttgc 480

tgctgctgct gccgctgctt tcagctctgg tctccggggc cactatggat gcccctaaaa 540

cttgcagccc taagcagttt gcctgcagag accaaatcac ctgtatctca aagggctggc 600

ggtgtgacgg tgaaagagat tgccccgacg gctctgatga agcccctgag atctgtccac 660

agagtaaagc ccagagatgc ccgccaaatg agcacagttg tctggggact gagctatgtg 720

tccccatgtc tcgtctctgc aacgggatcc aggactgcat ggatggctca gacgagggtg 780

ctcactgccg agagctccga gccaactgtt ctcgaatggg ttgtcaacac cattgtgtac 840

ctacacccag tgggcccacg tgctactgta acagcagctt ccagctgcag gcagatggca 900

agacgtgcaa agattttgac gagtgttccg tgtatggcac ctgcagccag ctttgcacca 960

acacagatgg ctccttcaca tgtggctgtg ttgaaggcta cctgctgcaa ccggacaacc 1020

gctcctgcaa ggccaagaat gagccagtag atcggccgcc agtgctactg attgccaact 1080

ctcagaacat cctagctacg tacctgagtg gggcccaagt gtctaccatc acacccacca 1140

gcacccgaca aaccacggcc atggacttca gttatgccaa tgagaccgta tgctgggtgc 1200

acgttgggga cagtgctgcc cagacacagc tcaagtgtgc ccggatgcct ggcctgaagg 1260

gctttgtgga tgagcatacc atcaacatct ccctcagcct gcaccacgtg gagcagatgg 1320

caatcgactg gctgacggga aacttctact ttgtcgacga cattgacgac aggatctttg 1380

tctgtaaccg aaacggggac acctgtgtca ctctgctgga cctggaactc tacaacccca 1440

aaggcatcgc cttggacccc gccatgggga aggtgttctt cactgactac gggcagatcc 1500

caaaggtgga gcgctgtgac atggatggac agaaccgcac caagctggtg gatagcaaga 1560

tcgtgtttcc acacggcatc accctggacc tggtcagccg cctcgtctac tgggcggacg 1620

cctacctaga ctacatcgag gtggtagact acgaagggaa gggtcggcag accatcatcc 1680

aaggcatcct gatcgagcac ctgtacggcc tgaccgtgtt tgagaactat ctctacgcca 1740

ccaactcgga caatgccaac acgcagcaga agacgagcgt gatccgagtg aaccggttca 1800

acagtactga gtaccaggtc gtcacccgtg tggacaaggg tggtgccctg catatctacc 1860

accagcgacg ccagccccga gtgcggagtc acgcctgtga gaatgaccag tacgggaagc 1920

caggtggctg ctccgacatc tgcctcctgg ccaacagtca caaggcaagg acctgcaggt 1980

gcaggtctgg cttcagcctg ggaagtgatg ggaagtcttg taagaaacct gaacatgagc 2040

tgttcctcgt gtatggcaag ggccgaccag gcatcattag aggcatggac atgggggcca 2100

aggtcccaga tgagcacatg atccccatcg agaaccttat gaatccacgc gctctggact 2160

tccacgccga gaccggcttc atctactttg ctgacaccac cagctacctc attggccgcc 2220

agaaaattga tggcacggag agagagacta tcctgaagga tggcatccac aatgtggagg 2280

gcgtagccgt ggactggatg ggagacaatc tttactggac tgatgatggc cccaagaaga 2340

ccattagtgt ggccaggctg gagaaagccg ctcagacccg gaagactcta attgagggca 2400

agatgacaca ccccagggcc attgtagtgg atccactcaa tgggtggatg tactggacag 2460

actgggagga ggaccccaag gacagtcggc gagggcggct cgagagggct tggatggacg 2520

gctcacaccg agatatcttt gtcacctcca agacagtgct ttggcccaat gggctaagcc 2580

tggatatccc agccggacgc ctctactggg tggatgcctt ctatgaccga attgagacca 2640

tactgctcaa tggcacagac cggaagattg tatatgaggg tcctgaactg aatcatgcct 2700

tcggcctgtg tcaccatggc aactacctct tttggaccga gtaccggagc ggcagcgtct 2760

accgcttgga acggggcgtg gcaggcgcac cgcccactgt gacccttctg cgcagcgaga 2820

gaccgcctat ctttgagatc cgaatgtacg acgcgcagca gcagcaagtg ggtaccaaca 2880

aatgccgggt aaataacgga ggctgcagca gcctgtgcct cgccaccccc gggagccgcc 2940

agtgtgcctg tgccgaggac caggtgttgg acacagatgg tgtcacctgc ttggcgaacc 3000

catcctacgt gcccccaccc cagtgccagc cgggcgagtt tgcctgtgcc aacaaccgct 3060

gcatccagga gcgctggaag tgtgacggag acaacgactg tctggacaac agcgatgagg 3120

ccccagcact gtgccatcaa cacacctgtc cctcggaccg attcaagtgt gagaacaacc 3180

ggtgtatccc caaccgctgg ctctgtgatg gggataatga ttgtggcaac agcgaggacg 3240

aatccaatgc cacgtgctca gcccgcacct gtccacccaa ccagttctcc tgtgccagtg 3300

gccgatgcat tcctatctca tggacctgtg atctggatga tgactgtggg gaccggtccg 3360

atgagtcagc ctcatgcgcc taccccacct gcttccccct gactcaattt acctgcaaca 3420

atggcagatg tattaacatc aactggcggt gtgacaacga caatgactgt ggggacaaca 3480

gcgacgaagc cggctgcagt cactcctgct ccagtaccca gttcaagtgc aacagtggca 3540

gatgcatccc cgagcactgg acgtgtgatg gggacaatga ttgtggggac tacagcgacg 3600

agacacacgc caactgtacc aaccaggcta caagacctcc tggtggctgc cactcggatg 3660

agttccagtg ccgcctagat ggcctgtgca tccccctgag gtggcgctgc gacggggaca 3720

ccgactgcat ggattccagc gatgagaaga gctgtgaggg cgtgacccat gtttgtgacc 3780

cgaatgtcaa gtttggctgc aaggactccg cccggtgcat cagcaaggcg tgggtgtgtg 3840

atggcgacag cgactgtgaa gataactccg acgaggagaa ctgtgaggcc ctggcctgca 3900

ggccaccctc ccatccctgc gccaacaaca cctctgtctg cctgcctcct gacaagctgt 3960

gcgacggcaa ggatgactgt ggagacggct cggatgaggg cgagctctgt gaccagtgtt 4020

ctctgaataa tggtggctgt agtcacaact gctcagtggc ccctggtgaa ggcatcgtgt 4080

gctcttgccc tctgggcatg gagctgggct ctgacaacca cacctgccag atccagagct 4140

actgtgccaa gcacctcaaa tgcagccaga agtgtgacca gaacaagttc agtgtgaagt 4200

gctcctgcta cgagggctgg gtcttggagc ctgacgggga aagctgccgc agtctggatc 4260

ccttcaaacc gttcatcatc ttctccaacc gccacgagat caggcgcatt gaccttcaca 4320

agggggacta cagcgtccta gtgcctggcc tgcgcaacac tattgccctg gacttccacc 4380

tcagccagag tgccctctac tggaccgacg tggtagagga caagatctac cgtgggaaac 4440

tcctggacaa cggagccctg accagctttg aggtggtgat tcagtatggc ttggccacac 4500

cagagggcct ggctgtagat tggattgcag gcaacatcta ctgggtggag agcaacctgg 4560

accagatcga agtggccaag ctggacggaa ccctccgaac cactctgctg gcgggtgaca 4620

ttgagcaccc gagggccatc gctctggacc ctcgggatgg gattctgttt tggacagact 4680

gggatgccag cctgccacga atcgaggctg cgtccatgag tggggctggc cgccgaacca 4740

tccaccggga gacaggctct gggggctggc ccaacgggct caccgtggat tacctggaga 4800

agcgcatcct ctggattgat gctaggtcag atgccatcta ttcagcccgg tatgacggct 4860

ccggccacat ggaggtgctt cggggacacg agttcctgtc acacccattt gccgtgacac 4920

tgtacggtgg ggaggtgtac tggaccgact ggcgaacaaa tacactggct aaggccaaca 4980

agtggactgg ccacaacgtc accgtggtac agaggaccaa cacccagccc ttcgacctgc 5040

aggtgtatca cccttcccgg cagcccatgg ctccaaaccc atgtgaggcc aatggcggcc 5100

ggggcccctg ttcccatctg tgcctcatca actacaaccg gaccgtctcc tgcgcctgtc 5160

cccacctcat gaagctgcac aaggacaaca ccacctgcta tgagtttaag aagttcctgc 5220

tgtacgcacg tcagatggag atccggggcg tggacctgga tgccccgtac tacaattata 5280

tcatctcctt cacggtgcct gatatcgaca atgtcacggt gctggactat gatgcccgag 5340

agcagcgagt ttactggtct gatgtgcgga ctcaagccat caaaagggca tttatcaacg 5400

gcactggcgt ggagaccgtt gtctctgcag acttgcccaa cgcccacggg ctggctgtgg 5460

actgggtctc ccgaaatctg ttttggacaa gttacgacac caacaagaag cagattaacg 5520

tggcccggct ggacggctcc ttcaagaatg cggtggtgca gggcctggag cagccccacg 5580

gcctggtcgt ccacccgctt cgtggcaagc tctactggac tgatggggac aacatcagca 5640

tggccaacat ggatgggagc aaccacactc tgctcttcag tggccagaag ggccctgtgg 5700

ggttggccat tgacttccct gagagcaaac tctactggat cagctctggg aaccacacaa 5760

tcaaccgttg caatctggat gggagcgagc tggaggtcat cgacaccatg cggagccagc 5820

tgggcaaggc cactgccctg gccatcatgg gggacaagct gtggtgggca gatcaggtgt 5880

cagagaagat gggcacgtgc aacaaagccg atggctctgg gtccgtggtg ctgcggaaca 5940

gtaccacgtt ggttatgcac atgaaggtgt atgacgagag catccagcta gagcatgagg 6000

gcaccaaccc ctgcagtgtc aacaacggag actgttccca gctctgcctg ccaacatcag 6060

agacgactcg ctcctgtatg tgtacagccg gttacagcct ccggagcgga cagcaggcct 6120

gtgagggtgt gggctctttt ctcctgtact ctgtacatga gggaattcgg gggattccac 6180

tagatcccaa tgacaagtcg gatgccctgg tcccagtgtc cggaacttca ctggctgtcg 6240

gaatcgactt ccatgccgaa aatgacacta tttattgggt ggatatgggc ctaagcacca 6300

tcagcagggc caagcgtgac cagacatggc gagaggatgt ggtgaccaac ggtattggcc 6360

gtgtggaggg catcgcggtg gactggatcg caggcaacat atactggacg gaccagggct 6420

tcgatgtcat cgaggttgcc cggctcaatg gctcttttcg ttatgtggtc atttcccagg 6480

gtctggacaa gcctcgggcc atcactgtcc acccagagaa ggggtacttg ttctggaccg 6540

agtggggtca ttacccacgt attgagcggt ctcgccttga tggcacagag agagtggtgt 6600

tggttaatgt cagcatcagc tggcccaatg gcatctcagt agactatcag ggcggcaagc 6660

tctactggtg tgatgctcgg atggacaaga tcgagcgcat cgacctggaa acgggcgaga 6720

accgggaggt ggtcctgtcc agcaataaca tggatatgtt ctccgtgtcc gtgtttgagg 6780

acttcatcta ctggagtgac agaactcacg ccaatggctc catcaagcgc ggctgcaaag 6840

acaatgctac agactccgtg cctctgagga caggcattgg tgttcagctt aaagacatca 6900

aggtcttcaa cagggacagg cagaagggta ccaatgtgtg cgcggtagcc aacggcgggt 6960

gccagcagct ctgcttgtat cggggtggcg gacagcgagc ctgtgcctgt gcccacggga 7020

tgctggcaga agacggggcc tcatgccgag agtacgctgg ctacctgctc tactcagagc 7080

ggaccatcct caagagcatc cacctgtcgg atgagcgtaa cctcaacgca ccggtgcagc 7140

cctttgaaga ccccgagcac atgaaaaatg tcatcgccct ggcctttgac taccgagcag 7200

gcacctcccc ggggacccct aaccgcatct tcttcagtga catccacttt gggaacatcc 7260

agcagatcaa tgacgatggc tcgggcagga ccaccatcgt ggaaaatgtg ggctctgtgg 7320

aaggcctggc ctatcaccgt ggctgggaca cactgtactg gacaagctac accacatcca 7380

ccatcacccg ccacaccgtg gaccagactc gcccaggggc cttcgagagg gagacagtca 7440

tcaccatgtc cggagacgac cacccgagag cctttgtgct ggatgagtgc cagaacctga 7500

tgttctggac caattggaac gagctccatc caagcatcat gcgggcagcc ctatccggag 7560

ccaacgtcct gaccctcatt gagaaggaca tccgcacgcc caatgggttg gccatcgacc 7620

accgggcgga gaagctgtac ttctcggatg ccaccttgga caagatcgag cgctgcgagt 7680

acgacggctc ccaccgctat gtgatcctaa agtcggagcc cgtccacccc tttgggttgg 7740

cggtgtacgg agagcacatt ttctggactg actgggtgcg gcgggctgtg cagcgagcca 7800

acaagtatgt gggcagcgac atgaagctgc ttcgggtgga cattccccag caacccatgg 7860

gcatcatcgc cgtggccaac gacaccaaca gctgtgaact ctccccctgc cgtatcaaca 7920

atggaggctg ccaggatctg tgtctgctca cccaccaagg ccacgtcaac tgttcctgtc 7980

gagggggccg gatcctccag gaggacttca cctgccgggc tgtgaactcc tcttgtcggg 8040

cacaagatga gtttgagtgt gccaatgggg aatgtatcag cttcagcctc acctgtgatg 8100

gcgtctccca ctgcaaggac aagtccgatg agaagccctc ctactgcaac tcacgccgct 8160

gcaagaagac tttccgccag tgtaacaatg gtcgctgtgt atccaacatg ctgtggtgca 8220

atggggtgga tgactgtggg gatggctctg atgagattcc ttgcaacaag actgcctgtg 8280

gtgtgggtga gttccgctgc cgggatgggt cctgcatcgg gaactccagt cgctgcaacc 8340

agtttgtgga ttgtgaggat gcctcggatg agatgaattg cagtaccaca gactgcagca 8400

gctatttccg cctgggcgtg aaaggtgtcc tcttccagcc gtgcgagcgg acatccctgt 8460

gctacgcacc tagctgggtg tgtgatggcg ccaacgactg tggagactac agcgatgaac 8520

gtgactgtcc aggtgtgaag cgccctaggt gcccgctcaa ttactttgcc tgccccagcg 8580

ggcgctgtat ccccatgagc tggacgtgtg acaaggagga tgactgtgag aacggcgagg 8640

acgagaccca ctgcaacaag ttctgctcag aggcacagtt cgagtgccag aaccaccggt 8700

gtatctccaa gcagtggctg tgtgacggta gcgatgattg cggggatggc tccgatgagg 8760

cagctcactg tgaaggcaag acatgtggcc cctcctcctt ctcctgtccc ggcacccacg 8820

tgtgtgtccc tgagcgctgg ctctgtgatg gcgacaagga ctgtaccgat ggcgcggatg 8880

agagtgtcac tgctggctgc ctgtacaaca gcacctgtga tgaccgtgag ttcatgtgcc 8940

agaaccgctt gtgtattccc aagcatttcg tgtgcgacca tgaccgtgac tgtgctgatg 9000

gctctgatga atcccctgag tgtgagtacc caacctgcgg ccccaatgaa ttccgctgtg 9060

ccaatgggcg ttgtctgagc tcccgtcagt gggaatgtga tggggagaat gactgtcacg 9120

accacagcga tgaggctccc aagaacccac actgcaccag cccagagcac aaatgcaatg 9180

cctcatcaca gttcctgtgc agcagcgggc gctgcgtggc tgaggcgttg ctctgcaacg 9240

gccaggacga ctgtggggac ggttcagacg aacgcgggtg ccatgtcaac gagtgtctca 9300

gccgcaagct cagtggctgc agtcaggact gcgaggacct caagataggc tttaagtgcc 9360

gctgtcgccc gggcttccgg ctaaaggacg atggcaggac ctgtgccgac ctggatgagt 9420

gcagcaccac cttcccctgc agccagctct gcatcaacac ccacggaagt tacaagtgtc 9480

tgtgtgtgga gggctatgca ccccgtggcg gtgaccccca cagctgcaaa gctgtgaccg 9540

atgaggagcc atttctcatc tttgccaacc ggtactacct gcggaagctc aacctggacg 9600

gctccaacta cacactgctt aagcagggcc tgaacaatgc ggtcgccttg gactttgact 9660

accgagagca gatgatctac tggacggacg tgaccaccca gggcagcatg attcgcagga 9720

tgcacctcaa cggcagcaac gtgcaggttc tgcaccggac gggccttagt aacccagatg 9780

ggctggctgt ggactgggtg ggtggcaacc tgtactggtg tgacaagggc agagatacca 9840

ttgaggtgtc caagcttaac ggggcctatc ggacagtgct ggtcagctct ggcctccggg 9900

agcccagagc tctggtagtg gatgtacaga atgggtacct gtactggaca gactggggtg 9960

accactcact gatcggccgg attggcatgg atggatctgg ccgcagcatc atcgtggaca 10020

ctaagatcac atggcccaat ggcctgaccg tggactacgt cacggaacgc atctactggg 10080

ctgacgcccg tgaggactac atcgagttcg ccagcctgga tggctccaac cgtcacgttg 10140

tgctgagcca agacatccca cacatctttg cgctgaccct atttgaagac tacgtctact 10200

ggacagactg ggaaacgaag tccatcaacc gggcccacaa gaccacgggt gccaacaaaa 10260

cactcctcat cagcaccctg caccggccca tggacttaca tgtattccac gccctgcgcc 10320

agccagatgt gcccaatcac ccctgcaaag tcaacaatgg tggctgcagc aacctgtgcc 10380

tgctgtcccc tgggggtggt cataaatgcg cctgccccac caacttctat ctgggtggcg 10440

atggccgtac ctgtgtgtcc aactgcacag caagccagtt tgtgtgcaaa aatgacaagt 10500

gcatcccctt ctggtggaag tgtgacacgg aggacgactg tggggatcac tcagacgagc 10560

ctccagactg tcccgagttc aagtgccgcc caggccagtt ccagtgctcc accggcatct 10620

gcaccaaccc tgccttcatc tgtgatgggg acaatgactg ccaagacaat agtgatgagg 10680

ccaattgcga cattcacgtc tgcttgccca gccaattcaa gtgcaccaac accaaccgct 10740

gcattcctgg catcttccgt tgcaatgggc aggacaactg cggggacggc gaggatgagc 10800

gggattgccc tgaggtgacc tgcgccccca accagttcca gtgctccatc accaagcgct 10860

gcatccctcg cgtctgggtc tgtgacaggg ataatgactg tgtggacggc agtgatgagc 10920

ctgccaactg tacccaaatg acctgtggag tggatgagtt ccgctgcaag gattctggcc 10980

gctgcatccc cgcgcgctgg aagtgtgacg gagaagatga ctgtggggat ggttcagatg 11040

agcccaagga agagtgtgat gagcgcacct gtgagccata ccagttccgc tgcaaaaaca 11100

accgctgtgt cccaggccgt tggcaatgtg actacgacaa cgactgcgga gataactcgg 11160

acgaggagag ctgcacacct cggccctgct ctgagagtga gttttcctgt gccaatggcc 11220

gctgcatcgc tgggcgctgg aagtgtgatg gggaccatga ctgtgccgac ggctcagacg 11280

agaaagactg caccccccgc tgtgatatgg accagttcca gtgcaagagt ggccactgca 11340

tccccctgcg ctggcgctgt gacgcggatg ctgactgtat ggacggcagt gacgaggaag 11400

cctgtggcac tggggtgagg acctgcccat tggatgagtt tcaatgtaac aacaccttgt 11460

gcaagccgct ggcctggaag tgtgatggag aggacgactg tggggacaac tcagatgaga 11520

accccgagga atgcgcccgg ttcatctgcc ctcccaaccg gcctttccgc tgcaagaatg 11580

accgagtctg cctgtggatt gggcgccagt gtgatggcgt ggacaactgt ggagatggga 11640

ctgacgagga ggactgtgag ccccccacgg cccagaaccc ccactgcaaa gacaagaagg 11700

agttcctgtg ccgaaaccag cgctgtctat catcctccct gcgctgtaac atgttcgatg 11760

actgcggcga tggctccgat gaagaagatt gcagcatcga ccccaagctg accagctgtg 11820

ccaccaatgc cagcatgtgt ggggacgaag ctcgttgtgt gcgcactgag aaagctgcct 11880

actgtgcctg ccgctcgggc ttccatactg tgccgggcca gcccggatgc caggacatca 11940

acgagtgcct gcgctttggt acctgctctc agctctgcaa caacaccaag ggaggccacc 12000

tctgcagctg tgcccgcaac ttcatgaaga cacacaacac ctgcaaagct gaaggctccg 12060

agtaccaggt gctatacatc gcggatgaca acgagatccg cagcttgttc ccgggccacc 12120

cccactcagc ctacgagcag acattccagg gcgatgagag tgtccgcata gatgccatgg 12180

atgtccatgt caaggccggc cgtgtctact ggactaactg gcacacgggc acaatctcct 12240

acaggagcct gccccctgcc gcccctccta ccacttccaa ccgccaccgg aggcagatcg 12300

accggggtgt cacccacctc aatatttcag ggctgaagat gccgaggggt atcgctatcg 12360

actgggtggc cgggaatgtg tactggaccg attccggccg agacgtgatt gaggtggcgc 12420

aaatgaaggg cgagaaccgc aagacgctca tctcgggcat gattgatgag ccccatgcca 12480

tcgtggtgga ccctctgagg ggcaccatgt actggtcaga ctgggggaac caccccaaga 12540

ttgaaacagc agcgatggat ggcacccttc gggagactct cgtgcaagac aacattcagt 12600

ggcctacagg gctggctgtg gactatcaca atgaacggct ctactgggca gatgccaagc 12660

tttcggtcat cggcagcatc cggctcaacg gcactgaccc cattgtggct gctgacagca 12720

aacgaggcct aagtcacccc ttcagcatcg atgtgtttga agactacatc tacggagtca 12780

cttacatcaa taatcgtgtc ttcaagatcc acaagtttgg acacagcccc ttgatcaacc 12840

taactggggg cctgagccat gcctctgatg tagtccttta ccatcaacac aagcagcctg 12900

aagtgaccaa cccctgtgac cgcaagaaat gtgaatggct gtgtctgctg agccccagcg 12960

ggcctgtctg cacctgtccc aatggaaaga ggctggataa tggcacctgt gtgcctgtgc 13020

cctctccaac accccctcca gatgccccta ggcctggaac ctgcactctg cagtgcttca 13080

atggtggtag ttgtttcctc aatgctcgga ggcagcccaa gtgccgttgc cagccccgtt 13140

acacaggcga taagtgtgag ctggatcagt gctgggaata ctgtcacaac ggaggcacct 13200

gtgcggcttc cccatctggc atgcccacgt gccgctgtcc cactggcttc acgggcccca 13260

aatgcaccgc acaggtgtgt gcaggctact gctctaacaa cagcacctgc accgtcaacc 13320

agggcaacca gccccagtgc cgatgtctac ctggcttcct gggcgaccgt tgccagtacc 13380

ggcagtgctc tggcttctgt gagaactttg gcacctgtca gatggctgct gatggctccc 13440

gacaatgtcg ctgcaccgtc tactttgagg gaccaaggtg tgaggtgaac aagtgtagtc 13500

gctgtctcca aggcgcctgt gtggtcaata agcagaccgg agatgtcaca tgcaactgca 13560

ctgatggccg ggtagccccc agttgtctga cctgcatcga tcactgtagc aatggtggct 13620

cctgcaccat gaacagcaag atgatgcctg agtgccagtg cccgccccat atgacaggac 13680

cccggtgcga ggagcaggtt gttagtcagc aacagcctgg gcatatggcc tccatcctga 13740

tccctctgct gctgcttctc ctgctgcttc tggtggctgg cgtggtgttc tggtataagc 13800

ggcgagtccg aggggctaag ggcttccagc accagcggat gaccaatggg gccatgaatg 13860

tggaaattgg aaaccctacc tacaagatgt atgaaggtgg agagcccgat gatgtcgggg 13920

gcctactgga tgctgatttt gcccttgacc ctgacaagcc taccaacttc accaacccag 13980

tgtatgccac gctctacatg gggggccacg gcagccgcca ttccctggcc agcacggacg 14040

agaagcgaga actgctgggc cggggacctg aagacgagat aggagatccc ttggcatagg 14100

gccctgcccc gacggatgtc cccagaaagc cccctgccac atgagtcttt caatgaaccc 14160

cctccccagc cggcccttct ccggccctgc cgggtgtaca aatgtaaaaa tgaaggaatt 14220

actttttata tgtgagcgag caagcgagca agcacagtat tatctctttg catttccttc 14280

ctgcctgctc ctcagtatcc cccccatgct gccttgaggg ggcggggagg gctttgtggc 14340

tcaaaggtat gaaggagtcc acatgttccc taccgagcat acccctggaa gctggcggca 14400

cggcctcccc accacgcctg tgcaagacac tcaacggggc tccgtgtccc agctttcctt 14460

tccttggctc tctggggtta gttcagggga ggtggagtcc tctgctgacc ctgtctggaa 14520

gatttggctc tagctgagga aggagtcttt tagttgaggg aagtcacccc aaaccccagc 14580

tcccactttc aggggcacct ctcagatggc catgctcagt atcccttcca gacaggccct 14640

cccctctcta gcgccccctc tgtggctcct agggctgaac acattctttg gtaactgtcc 14700

cccaagcctc ccatccccct gagggccagg aagagtcggg gcacaccaag gaagggcaag 14760

cgggcagccc cattttgggg acgtgaacgt tttaataatt tttgctgaat tcctttacaa 14820

ctaaataaca cagatattgt tataaataaa attgtaaaaa aggaaaaaaa aaagaaaaga 14880

aaagaaaaga aaaaaaaaaa aaaaaaa 14907

<210> 5

<211> 5265

<212> DNA

＜213〉homo sapiens

<300>

<308> NM_000527

<309> 2010-08-01

<313> (1)..(5265)

<400> 5

acatttgaaa atcaccccac tgcaaactcc tccccctgct agaaacctca cattgaaatg 60

ctgtaaatga cgtgggcccc gagtgcaatc gcgggaagcc agggtttcca gctaggacac 120

agcaggtcgt gatccgggtc gggacactgc ctggcagagg ctgcgagcat ggggccctgg 180

ggctggaaat tgcgctggac cgtcgccttg ctcctcgccg cggcggggac tgcagtgggc 240

gacagatgcg aaagaaacga gttccagtgc caagacggga aatgcatctc ctacaagtgg 300

gtctgcgatg gcagcgctga gtgccaggat ggctctgatg agtcccagga gacgtgcttg 360

tctgtcacct gcaaatccgg ggacttcagc tgtgggggcc gtgtcaaccg ctgcattcct 420

cagttctgga ggtgcgatgg ccaagtggac tgcgacaacg gctcagacga gcaaggctgt 480

ccccccaaga cgtgctccca ggacgagttt cgctgccacg atgggaagtg catctctcgg 540

cagttcgtct gtgactcaga ccgggactgc ttggacggct cagacgaggc ctcctgcccg 600

gtgctcacct gtggtcccgc cagcttccag tgcaacagct ccacctgcat cccccagctg 660

tgggcctgcg acaacgaccc cgactgcgaa gatggctcgg atgagtggcc gcagcgctgt 720

aggggtcttt acgtgttcca aggggacagt agcccctgct cggccttcga gttccactgc 780

ctaagtggcg agtgcatcca ctccagctgg cgctgtgatg gtggccccga ctgcaaggac 840

aaatctgacg aggaaaactg cgctgtggcc acctgtcgcc ctgacgaatt ccagtgctct 900

gatggaaact gcatccatgg cagccggcag tgtgaccggg aatatgactg caaggacatg 960

agcgatgaag ttggctgcgt taatgtgaca ctctgcgagg gacccaacaa gttcaagtgt 1020

cacagcggcg aatgcatcac cctggacaaa gtctgcaaca tggctagaga ctgccgggac 1080

tggtcagatg aacccatcaa agagtgcggg accaacgaat gcttggacaa caacggcggc 1140

tgttcccacg tctgcaatga ccttaagatc ggctacgagt gcctgtgccc cgacggcttc 1200

cagctggtgg cccagcgaag atgcgaagat atcgatgagt gtcaggatcc cgacacctgc 1260

agccagctct gcgtgaacct ggagggtggc tacaagtgcc agtgtgagga aggcttccag 1320

ctggaccccc acacgaaggc ctgcaaggct gtgggctcca tcgcctacct cttcttcacc 1380

aaccggcacg aggtcaggaa gatgacgctg gaccggagcg agtacaccag cctcatcccc 1440

aacctgagga acgtggtcgc tctggacacg gaggtggcca gcaatagaat ctactggtct 1500

gacctgtccc agagaatgat ctgcagcacc cagcttgaca gagcccacgg cgtctcttcc 1560

tatgacaccg tcatcagcag agacatccag gcccccgacg ggctggctgt ggactggatc 1620

cacagcaaca tctactggac cgactctgtc ctgggcactg tctctgttgc ggataccaag 1680

ggcgtgaaga ggaaaacgtt attcagggag aacggctcca agccaagggc catcgtggtg 1740

gatcctgttc atggcttcat gtactggact gactggggaa ctcccgccaa gatcaagaaa 1800

gggggcctga atggtgtgga catctactcg ctggtgactg aaaacattca gtggcccaat 1860

ggcatcaccc tagatctcct cagtggccgc ctctactggg ttgactccaa acttcactcc 1920

atctcaagca tcgatgtcaa cgggggcaac cggaagacca tcttggagga tgaaaagagg 1980

ctggcccacc ccttctcctt ggccgtcttt gaggacaaag tattttggac agatatcatc 2040

aacgaagcca ttttcagtgc caaccgcctc acaggttccg atgtcaactt gttggctgaa 2100

aacctactgt ccccagagga tatggttctc ttccacaacc tcacccagcc aagaggagtg 2160

aactggtgtg agaggaccac cctgagcaat ggcggctgcc agtatctgtg cctccctgcc 2220

ccgcagatca acccccactc gcccaagttt acctgcgcct gcccggacgg catgctgctg 2280

gccagggaca tgaggagctg cctcacagag gctgaggctg cagtggccac ccaggagaca 2340

tccaccgtca ggctaaaggt cagctccaca gccgtaagga cacagcacac aaccacccga 2400

cctgttcccg acacctcccg gctgcctggg gccacccctg ggctcaccac ggtggagata 2460

gtgacaatgt ctcaccaagc tctgggcgac gttgctggca gaggaaatga gaagaagccc 2520

agtagcgtga gggctctgtc cattgtcctc cccatcgtgc tcctcgtctt cctttgcctg 2580

ggggtcttcc ttctatggaa gaactggcgg cttaagaaca tcaacagcat caactttgac 2640

aaccccgtct atcagaagac cacagaggat gaggtccaca tttgccacaa ccaggacggc 2700

tacagctacc cctcgagaca gatggtcagt ctggaggatg acgtggcgtg aacatctgcc 2760

tggagtcccg tccctgccca gaacccttcc tgagacctcg ccggccttgt tttattcaaa 2820

gacagagaag accaaagcat tgcctgccag agctttgttt tatatattta ttcatctggg 2880

aggcagaaca ggcttcggac agtgcccatg caatggcttg ggttgggatt ttggtttctt 2940

cctttcctcg tgaaggataa gagaaacagg cccgggggga ccaggatgac acctccattt 3000

ctctccagga agttttgagt ttctctccac cgtgacacaa tcctcaaaca tggaagatga 3060

aaggggaggg gatgtcaggc ccagagaagc aagtggcttt caacacacaa cagcagatgg 3120

caccaacggg accccctggc cctgcctcat ccaccaatct ctaagccaaa cccctaaact 3180

caggagtcaa cgtgtttacc tcttctatgc aagccttgct agacagccag gttagccttt 3240

gccctgtcac ccccgaatca tgacccaccc agtgtctttc gaggtgggtt tgtaccttcc 3300

ttaagccagg aaagggattc atggcgtcgg aaatgatctg gctgaatccg tggtggcacc 3360

gagaccaaac tcattcacca aatgatgcca cttcccagag gcagagcctg agtcactggt 3420

cacccttaat atttattaag tgcctgagac acccggttac cttggccgtg aggacacgtg 3480

gcctgcaccc aggtgtggct gtcaggacac cagcctggtg cccatcctcc cgacccctac 3540

ccacttccat tcccgtggtc tccttgcact ttctcagttc agagttgtac actgtgtaca 3600

tttggcattt gtgttattat tttgcactgt tttctgtcgt gtgtgttggg atgggatccc 3660

aggccaggga aagcccgtgt caatgaatgc cggggacaga gaggggcagg ttgaccggga 3720

cttcaaagcc gtgatcgtga atatcgagaa ctgccattgt cgtctttatg tccgcccacc 3780

tagtgcttcc acttctatgc aaatgcctcc aagccattca cttccccaat cttgtcgttg 3840

atgggtatgt gtttaaaaca tgcacggtga ggccgggcgc agtggctcac gcctgtaatc 3900

ccagcacttt gggaggccga ggcgggtgga tcatgaggtc aggagatcga gaccatcctg 3960

gctaacacgt gaaaccccgt ctctactaaa aatacaaaaa attagccggg cgtggtggcg 4020

ggcacctgta gtcccagcta ctcgggaggc tgaggcagga gaatggtgtg aacccgggaa 4080

gcggagcttg cagtgagccg agattgcgcc actgcagtcc gcagtctggc ctgggcgaca 4140

gagcgagact ccgtctcaaa aaaaaaaaac aaaaaaaaac catgcatggt gcatcagcag 4200

cccatggcct ctggccaggc atggcgaggc tgaggtggga ggatggtttg agctcaggca 4260

tttgaggctg tcgtgagcta tgattatgcc actgctttcc agcctgggca acatagtaag 4320

accccatctc ttaaaaaatg aatttggcca gacacaggtg cctcacgcct gtaatcccag 4380

cactttggga ggctgagctg gatcacttga gttcaggagt tggagaccag gcctgagcaa 4440

caaagcgaga tcccatctct acaaaaacca aaaagttaaa aatcagctgg gtacggtggc 4500

acgtgcctgt gatcccagct acttgggagg ctgaggcagg aggatcgcct gagcccagga 4560

ggtggaggtt gcagtgagcc atgatcgagc cactgcactc cagcctgggc aacagatgaa 4620

gaccctattt cagaaataca actataaaaa aataaataaa tcctccagtc tggatcgttt 4680

gacgggactt caggttcttt ctgaaatcgc cgtgttactg ttgcactgat gtccggagag 4740

acagtgacag cctccgtcag actcccgcgt gaagatgtca caagggattg gcaattgtcc 4800

ccagggacaa aacactgtgt cccccccagt gcagggaacc gtgataagcc tttctggttt 4860

cggagcacgt aaatgcgtcc ctgtacagat agtggggatt ttttgttatg tttgcacttt 4920

gtatattggt tgaaactgtt atcacttata tatatatata tacacacata tatataaaat 4980

ctatttattt ttgcaaaccc tggttgctgt atttgttcag tgactattct cggggccctg 5040

tgtagggggt tattgcctct gaaatgcctc ttctttatgt acaaagatta tttgcacgaa 5100

ctggactgtg tgcaacgctt tttgggagaa tgatgtcccc gttgtatgta tgagtggctt 5160

ctgggagatg ggtgtcactt tttaaaccac tgtatagaag gtttttgtag cctgaatgtc 5220

ttactgtgat caattaaatt tcttaaatga accaatttgt ctaaa 5265

<210> 6

<211> 1223

<212> DNA

＜213〉homo sapiens

<300>

<308> NM_000041.2

<309> 2010-08-15

<313> (1)..(1223)

<400> 6

gggatccttg agtcctactc agccccagcg gaggtgaagg acgtccttcc ccaggagccg 60

actggccaat cacaggcagg aagatgaagg ttctgtgggc tgcgttgctg gtcacattcc 120

tggcaggatg ccaggccaag gtggagcaag cggtggagac agagccggag cccgagctgc 180

gccagcagac cgagtggcag agcggccagc gctgggaact ggcactgggt cgcttttggg 240

attacctgcg ctgggtgcag acactgtctg agcaggtgca ggaggagctg ctcagctccc 300

aggtcaccca ggaactgagg gcgctgatgg acgagaccat gaaggagttg aaggcctaca 360

aatcggaact ggaggaacaa ctgaccccgg tggcggagga gacgcgggca cggctgtcca 420

aggagctgca ggcggcgcag gcccggctgg gcgcggacat ggaggacgtg tgcggccgcc 480

tggtgcagta ccgcggcgag gtgcaggcca tgctcggcca gagcaccgag gagctgcggg 540

tgcgcctcgc ctcccacctg cgcaagctgc gtaagcggct cctccgcgat gccgatgacc 600

tgcagaagcg cctggcagtg taccaggccg gggcccgcga gggcgccgag cgcggcctca 660

gcgccatccg cgagcgcctg gggcccctgg tggaacaggg ccgcgtgcgg gccgccactg 720

tgggctccct ggccggccag ccgctacagg agcgggccca ggcctggggc gagcggctgc 780

gcgcgcggat ggaggagatg ggcagccgga cccgcgaccg cctggacgag gtgaaggagc 840

aggtggcgga ggtgcgcgcc aagctggagg agcaggccca gcagatacgc ctgcaggccg 900

aggccttcca ggcccgcctc aagagctggt tcgagcccct ggtggaagac atgcagcgcc 960

agtgggccgg gctggtggag aaggtgcagg ctgccgtggg caccagcgcc gcccctgtgc 1020

ccagcgacaa tcactgaacg ccgaagcctg cagccatgcg accccacgcc accccgtgcc 1080

tcctgcctcc gcgcagcctg cagcgggaga ccctgtcccc gccccagccg tcctcctggg 1140

gtggacccta gtttaataaa gattcaccaa gtttcacgca aaaaaaaaaa aaaaaaaaaa 1200

aaaaaaaaaa aaaaaaaaaa aaa 1223

<210> 7

<211> 897

<212> DNA

＜213〉homo sapiens

<300>

<308> NM_000039

<309> 2010-08-15

<313> (1)..(897)

<400> 7

agagactgcg agaaggaggt cccccacggc ccttcaggat gaaagctgcg gtgctgacct 60

tggccgtgct cttcctgacg gggagccagg ctcggcattt ctggcagcaa gatgaacccc 120

cccagagccc ctgggatcga gtgaaggacc tggccactgt gtacgtggat gtgctcaaag 180

acagcggcag agactatgtg tcccagtttg aaggctccgc cttgggaaaa cagctaaacc 240

taaagctcct tgacaactgg gacagcgtga cctccacctt cagcaagctg cgcgaacagc 300

tcggccctgt gacccaggag ttctgggata acctggaaaa ggagacagag ggcctgaggc 360

aggagatgag caaggatctg gaggaggtga aggccaaggt gcagccctac ctggacgact 420

tccagaagaa gtggcaggag gagatggagc tctaccgcca gaaggtggag ccgctgcgcg 480

cagagctcca agagggcgcg cgccagaagc tgcacgagct gcaagagaag ctgagcccac 540

tgggcgagga gatgcgcgac cgcgcgcgcg cccatgtgga cgcgctgcgc acgcatctgg 600

ccccctacag cgacgagctg cgccagcgct tggccgcgcg ccttgaggct ctcaaggaga 660

acggcggcgc cagactggcc gagtaccacg ccaaggccac cgagcatctg agcacgctca 720

gcgagaaggc caagcccgcg ctcgaggacc tccgccaagg cctgctgccc gtgctggaga 780

gcttcaaggt cagcttcctg agcgctctcg aggagtacac taagaagctc aacacccagt 840

gaggcgcccg ccgccgcccc ccttcccggt gctcagaata aacgtttcca aagtggg 897

<210> 8

<211> 1299

<212> DNA

＜213〉homo sapiens

<400> 8

acgcccggga acccccgacc cctctgagcc cggggtactg cgcccgggtc tccacgccca 60

gagatgctcc ccggtctcca ccgtcgggca agccccaagc gcagcagcgc agagtcctgg 120

ggtcaccaga gctcgtacta ggacatcgtc tccccattta acaccgcctc cggtcccatc 180

tgagttgcaa gtggtgggga tgtggggctc cggatcaaag tccccgaaac cgagcacttc 240

ccgaagcctc cttggcctcg aaacaaaaca ataacgccca actccatcat attccagaac 300

tcccaccacc tgcatacaga cattcagctg cacaagcccc ctccatgcta cagtcaacag 360

gatctccagg ccacggctca agcccaggta ctcacatcag tggttctatc aacactcagg 420

acagacccat agaagaggcc caagcaggcc ctggaagtgc atgtggaggc caccaggcaa 480

ggaattctgg agtcccaggt actcataact ctgggtggca tggccccttt gcaccatgga 540

ctgtttgccc ttagaaaggg atggatctga gctgggcgca gtggctcatg cctgtaatcc 600

cagcactttg ggaggccaag gtgggcagct cacctcaggt caggttggtc tcaaactcct 660

gacctcaggc gatccacctc agcctctcaa agtgctggaa ttataggtgt gagccactgt 720

gcccagccca aaatcattct ttttggaatt ttgaagcata taattccaaa aggtatgaag 780

gtaatcactt agattgctct aataagggaa tgggaacagt taagtcctat acaaataaga 840

caaagataag atactacaaa aaggggatga gcccaagaaa aaaatcaaag tcccagagag 900

agaacagcca ttgattctaa atacacaagt ctatggcccc aacccaaact tgtttcacta 960

agaacaacct gtggtttcga gaatctggtc atcccccaca gtgaatacat gaacacattg 1020

taatgtttga aatgtttatt tttcttgttg atttcttact gttagaagag ctaagtgatt 1080

tggcccaaag tggctaagtg attcggccag tttgtacaca gggatataag tttgctgaca 1140

ccaagctcat actttacaaa tgtaatatct tcataaaaca aaaatactgg gccgggcgcg 1200

gtggctcacg cctgtaatcc cagcattttg ggaggccgag gcgggcggat catgagatca 1260

ggagatcgag accatcctgg ctagcagggt gaagccccg 1299

<210> 9

<211> 917

<212> DNA

＜213〉house mouse

<400> 9

caaaatggcg tgctaccctg tccaaccttg tctgtagaca gagtcaattg aacactgtct 60

ttgggacttc cgtgcaactg aggtgggcgg gcttgaagca caaagctttc agggagaacc 120

aaactttatg cccaagctgc tctctgccac ccacagggta aatgaatctc atacaggaaa 180

ggcaagagac atgtgacact gttgtctgat ggtcacaagt caagcttttt aaaaagcagc 240

ctgatattgt gagctaacat ggctttctgt aattgaatgc aatgtatttt ctatgcttgt 300

ctgggtaaag ttgaccttgg tttgatttag ctcaagcaat atttcaacag tgcactgggg 360

ctctgagtcc cctgactact gtttgactag agccaggctc tgccctggat ggcaaccaac 420

agcccaggct ctggggcaca gccgggcttt gacaggtctg gggaaatgtt caccggagat 480

gaaaggtttc aaactatgaa actctaaaat ctcaagtcaa aacttttgac aagcacacac 540

aggacatgaa ttacaatcac ccgaagattt ttacaggctt ctcaatttta atgacatgct 600

gacacgtgtc atcagatctc acaacaagat gacacatggg tgtcaggtat ggcgcagaag 660

actagagtcg gggtgtaacc aatgagcatt gtctgttgga cacaggcgaa ttcggcaaac 720

ggacagtgct ggaggcagaa gggtttaaag aaggcaggaa agcccatgtt taacagaatg 780

gggtgacgaa gagggatggg aaggtctaac tcacccgggg gtgggggcac caggggggcc 840

cacggacaca gaaaaccacg caggtcaggc acctacaaag accgaaggaa aaagggacca 900

cgcagaaatc actcacg 917

<210> 10

<211> 1550

<212> DNA

＜213〉homo sapiens

<400> 10

aaagtccaag gtgggaacag ataggtctgg gggcatgggg gctgggccta ataggggccg 60

ggcatggatg ggcctctcct gctcaccgat cctgggctgg gcatcttgtc cttggtgggt 120

gggggccaag gaaggagtgg tggggcttgg cccagagtct ggtgtggctg tgactgacca 180

cagcttgtga tgccccagcc aagacctcag gcacaccccg tcccccactc cgccccccct 240

gggttagaca actgagagtc acagtgtggt gggagaaggg acgtcattcc tctaagggac 300

aagcttttgg cccctcccca caccagggca ggtacttatg tcggggctta tgcagggcag 360

aagggctttg gccaggtcag ctgccagggg ctggggccca ggctccccag ggtctggcgt 420

ggtgcatcag gggcctggtg ggggcttacc gaggatgacg ggccgtgtgt ggttactgag 480

ctcagccttg ggcgtggtgt gcggggggaa gagcacattg aggagccaga agggggcggc 540

aggagggagc agcagcccca gcagcagcgt cacccactgc catggggagc cgggcggccc 600

cattccagcc ctggtgccta ctgccagaga aagcggcact gggctgtcct tatctggtgt 660

gggagtggga ggggccctag ggccagtggg agggacggcc tggccagtgg gggttgtggc 720

cagagattgc cggaaagggg cacagcctca gggagccggc tctgggcctg ggttcagttc 780

cgccttcttc tcttggcgcc aggggaaaca gagccggggc agcaggaggc ccagaactac 840

acaatgtttt attgaaaaag tcaggcctca gctcagctgt ctccattcgg ctcagcttgg 900

tggggggccc tgcccatagt agactgagcc agatcttcct gcaggcagct gggctggact 960

ccctccctgg ctacccttcc cttcgtctct gatggtgaca tccaaacaat aaatatgcaa 1020

taaatagcgc tcctgggctg ggccgggccg gctgccttca aaccccactc ggcccctacc 1080

agtcttctct ggccaggaca ggcctactgg ggtgctagat agtaaagtcc ccaaacatcc 1140

cagggtccca caagacctgg gatccatctc cattttgagg cccaggcctg gtttccaagg 1200

agacctagca aagctgggtc caggacaggg ccaggcaagc agggctggca ggtgggtgct 1260

gggaagaggc tgttacccca gaccacagct tgttatgtcc tggccaagac ctcgactcca 1320

ggccacaaga tgacactggg ctcaggagta gatggtgatg acttcacggc caccaccgcg 1380

caccaacagc acccgctcaa ggccctcggt cagcacctcg aggaactcca tgtagttctc 1440

gtcgccctca ctgttcctgg gtgggccagg catgtttagg agaaccaggc gggcgtcgtg 1500

ggagcgcgtg acaatgactt cattgagctt cacagcagtg tgcatgcgcc 1550

<210> 11

<211> 329

<212> DNA

＜213〉homo sapiens

<220>

<221> misc_feature

<222> (6)..(6)

＜223〉n is a, c, g or t

<220>

<221> misc_feature

<222> (134)..(134)

＜223〉n is a, c, g or t

<220>

<221> misc_feature

<222> (311)..(311)

＜223〉n is a, c, g or t

<220>

<221> misc_feature

<222> (327)..(327)

＜223〉n is a, c, g or t

<400> 11

tttaangttt gatgtttatt ggtggtgtct gatgagcgtt tctcttgtcc agactgtgtt 60

tctctctcca gaccagctcc cagggtacag ggggtgggga gtaggtggta gctgtgtcag 120

tgctgggccc tggngccact ccctagggaa gagcaggtgg ggcctcgggg ggtctggccc 180

tagctctggc agatccatcc tcagtgaagc acatccctgg ggcaaaggca ctcctgaggc 240

caagaccagc atgggcttga tggagccacc ccagggagcc ccaagagaga tgaagccatc 300

aataaagcgg nccttccagg cctgggnct 329

<210> 12

<211> 1826

<212> DNA

＜213〉homo sapiens

<400> 12

tgtcactctg acctcagtgt aggcactgcc tcctctggga agtctttgct gacctgaaag 60

gctcagcctc ttgtgcttcc taagcttttc tcagagcatt tagcttcatt agtaattaaa 120

cttccattag tgaaatgatc tgattaatgg ttgtcactcc cagattttaa ttctaacttt 180

tttttttttt tttttttttg agacccagtc tctttttttt tgagacagtc tcattctgcc 240

gcccagtctg gagtgcaacg acgtgatctc ggctcacggt gacctccacc tcccaggttc 300

aagtgattct cgtgcctcag cctcctgagt agctgggacg acagatgcat gccaccacgc 360

ctggcaaata ttttgtattt tagtagagac gggggtttct gccgtgttgg cctggctggt 420

ctcaaactcc tgagttcggg tgatccgcct gcctcggtct cccggggtgc cgggattaca 480

ggcgtgagcc accgtgcccg gcctctaaac acttgtggcc ctgtcattca cccagcactc 540

aaaaggtcgt ctcacctgcc cttttgggag ctgggagaga cagctcaaat tgtcaccgcc 600

cccccaccgc cccgtgctcc tctgacaggg ctgtgggtgg agccagctcc agtccccgcg 660

cccagcacag aggcaggcac ggtgcacact gcctcaacag ctcgaccagg agagtgggca 720

gctgtacatc tagggtgccc agctcagtcc caggcctcag cagagcccat cttgcctcac 780

tgcacacagc actgagcctg tggctggtga ggagtgaaac ctagtgtggg actctagtgc 840

ctcccttcaa cctgaaacat agccatcagg gcttacggta gcaaaggaag gtctttattc 900

aggaggcggg ggctctgggc tggcagtcgg ggatgcaggg ggaccctggc ggtaggcacc 960

cagcaggatg gcattgatgt gctccagggt caggttgctg aagaccatgt tcagatgctg 1020

tatcccgtgc aggggcagca ggtgcacagg ctgtggctgg cggccctgcc acaggccaca 1080

gagctcggtg ctgcgggtcg ccaccgtgtc atcaccatcc tcatagagca cacccacagg 1140

gtccgtgtag gggaagccgt ggtcgtagat gtaggtgcgg ggcgtgggca ggcccacgcc 1200

gtaaagacag tatacttcca caccaggtgc tgggagtcct gccaggaggt cacgtgactg 1260

cagccacatg taccagcctt cctcaaagtg caggtctgca aagaagcgtt ggaagtcacg 1320

gcctgtgtag ttgaagctgg gtgtggaaat gaacacgtgg tcctcaggcc acgccatgcg 1380

agagggaaac atccaggggg aggtggtggt tatgcgctgc tcctctttca gcttgatgct 1440

ggacatgatg gggatgccct ggttgtcacc tgtggatatg gagcaaggtg ggacagggag 1500

ccaggcctgg ctacccctgg cccacaacct gctgagtgta ggctcagcca gatgctcaat 1560

cttgtccctg cccaatctag acacagactc taagccacag gcttgagcag gcctgatatt 1620

caatgatgct cagtgtcagc ttactcaatg agaagccctg ataagacctc tgttgggtgg 1680

agctgtaggg cttcaaaagg atggcaggga caggcaccat ggctcacccc tgtaatcccg 1740

gcactttggg aggctgaggc aggaggatca cttgaggcca ggagtccgtg accagactgg 1800

gcaatgcagt gagaccctgt ctctac 1826

<210> 13

<211> 536

<212> DNA

＜213〉homo sapiens

<400> 13

tgtttttttt tttttttttt aaagagatgg agtcctgatc tgtcgcccag gctggagttc 60

agtggcacaa tattggccca ctgcaaccct tgaaccttcc cggttcaagt gattctcctg 120

cctaggtctt ctgagtagct gggattacag gtgcccacca ccacgcccag ctaatttttg 180

tggttttagt agagacggag ttttgccatg ttggccaagc tagtctcaaa ctcctgacct 240

caagtgatcg gccggcctca gcctcccaaa gtgctgggat tacaggcttg agccactgcg 300

cctggcccag ttttcccatg tcttgaggca ccactaccca tgcacctcag aatcctccct 360

tgcctttatc cctttgatac agcacatccc aaagtgaatc cccacatggt ccctggttgc 420

tcagactcag tgaaaaaaaa atgaatggtc aagtgagttt tggaaaaccc caaacgcttg 480

aaaaattctt ggcacacata aacatattca aggctctgag aagttctgca gcacaa 536

<210> 14

<211> 551

<212> DNA

＜213〉homo sapiens

<400> 14

accattcagc tgcacccaga tgccccaaga gcaatgagcc cacacgcaga gctggaggac 60

ctgaaaggca acctccaagt cccagatcat gtctctgtgg ggtctggtct ccaagatgcc 120

cccagaaaaa gtgcagcggc tctatgtcga ctttccccaa cacctgcggc atcttctggg 180

tgactggctg gagagccagc cctgggagtt cctggtcggc tccgacgcct tctgctgcaa 240

cttggctagt gccctacttt cagacactgt ccagcacctt caggcctcgg tgggagagca 300

gggggagggg agcaccatct tgcaacacat cagcaccctt gagagcatat atcagaggga 360

ccccctgaag ctggtggcca ctttcagaca aatacttcaa ggagagaaaa aagctgttat 420

ggaacagttc cgccacttgc caatgccttt ccactggaag caggaagaac tcaagtttaa 480

gacaggcttg cggaggctgc agcaccgagt aggggagatc caccttctcc gagaagccct 540

gcagaagggg g 551

<210> 15

<211> 672

<212> DNA

＜213〉house mouse

<400> 15

tgtgacaccc ttcctcacgc ttagacagca aagttgcctc ggaagagaag agaagcctgc 60

atgggaatgg ccagcacatc ctaaatgctc cagtggcccg tggttcgtcc cttcgtctca 120

ttgactgcca cagacaggaa gtaggctcag ggacttggca cctacccaac agcaggacgt 180

cctttctggc catactcctg agggtaacaa aatcacatgg aagcccaaag caagccaggc 240

tcaggctcct ctgccctctg ctacttaaca atgtccgtcc ttccccagcc cccctgcaga 300

tgcttctgta tgggaaagcc cctctgcatc taatgacact ctgctttcaa agacgggaca 360

gtccctggtc tctggagagt gaccattcgt ggccttctca gttgacactt ctccgctgag 420

gcatccctta gccctgaacc agaaatgaaa gagccggctc agagtgaaaa ggaagaatag 480

ccatcaatct gctcctgtgt gcaaggagca cagacctggt ctcagactct gcccgtctcc 540

cccgcttccc tgccctctga gtgactcacg gtgcaggctg agggagatgt tgatggtatg 600

ctcatccaca aagcccttca ggccaggcat ccgggcaact tgagctgtgt ctgggcacac 660

tgtcccaacg tg 672

<210> 16

<211> 616

<212> DNA

＜213〉house mouse

<400> 16

gacttttatc tgcactgttt caacagcagg tagccagccg tctttttact gcctgcctct 60

ggctgaagct cggcccacac tatcaggact cagccctgta gggatgactc tgccacacag 120

ctacagcacc agctggcaca aatggctttc tctccaactt cctcaggctt ccctgagtca 180

ctgcccagcc ctaggactgg caacaccctg gccctgctca cccatccacc cttggcaaga 240

gggaaagagg aagaagcctg cagagagctg gtgccctgct tccagatgct gctccattct 300

caggccaagc ctcaagatgg ggggaacctg agtgggagcc tctctcctgg cttgcgttcc 360

ctcccacttc tgggaaagca gggcagtgac agtccctgtt ctcatgtgtc tgcccttggc 420

tgggctcccc tcacctcccc aaagaccagg cagggtccca ttcagcagac ctgactgtaa 480

ggaattggca agaaatgacg tccctagcca gcctggcctc ccctttggta tttttgcagc 540

tggagattat tagtctcaag caaaactcct ttgttatcca agcccactcc accacattat 600

tttcctctct cctaaa 616

<210> 17

<211> 471

<212> DNA

＜213〉house mouse

<400> 17

gacagggtct tattctgcag ctcgggttga cctggaattc tccaggcaga ccattctggc 60

cttacgttca ctgacatcca cctgcctctg cctcctgagt gctgctgtta aggagtagtt 120

ccagcctata gtgttctgaa atactgttat tttactgtaa tgatagccaa agctaaaatg 180

agttaagaat agttcctttc ttactcgctg tctcgttcct cattcacttg ccccatcttc 240

gtgccctcag aactacccca cccccaatcc tcctttagcc ccagagcctt ctctgaaccc 300

taccccttgc ttcctgtcag catctcaggg ccccctcttt gcttccttaa tctctactgg 360

aaacacagag aactcccctg ccctctgcca ttcttctgct ggagctacct tcccaccctt 420

gtgcaagcca ggcccctcat acccaagcag gtgacaccat ctgtgtccaa c 471

<210> 18

<211> 707

<212> DNA

＜213〉homo sapiens

<400> 18

gagctgcgct caccgctgtt gcctgatttt ggtctaagtg aaggcttgcg gttcagattc 60

caacaacttc cctttgtaaa ggaaatggac aagaaactcc cccctggata tgccttgaag 120

ccagctacag cgtgaggtgg tgcagctaga aagtgctaga aacacacacc agctctcaga 180

agtctggagg aaaacatcag gggtgtagtc tccttgacaa cagaggaaac atcacattct 240

cagccatccc gggagagaga aactaaagtg atgaacaaac aaggccttgc ctaagacttc 300

cttaacattt tctcttaagg aagaggttga ttgaggaaaa atcgccgctt ggacagctga 360

accgaagcca ttcacagcct ctgaagaagc gaggccaccc cagggggtcg gtcccggggg 420

atagctgccc caccgtggct gaagatctcg gctgcagacc aaggaggggc ggggagattc 480

tgaggcaacg tttcaacctc gtaaggaacc gaggccttga gggtggccgg gggcccctct 540

gtgaacttga tcggggctgg tggggcgagg gcgcccccca ggacaaggtg gggacggagg 600

tgtcgccaca gagcacagcg gaaaccgggg acttcgccag gagggcccag gataacggag 660

ggcgactcgt gtatgtcgcg gaggcggctc cggggacccg ggacttg 707

<210> 19

<211> 741

<212> DNA

＜213〉homo sapiens

<400> 19

ggggctccct ctcaacctat tctggcgcct ggagcaagcc ttacctgcag tccccgccgc 60

ggcgaggagc aaggcgacgg tccagcgcaa tttccagccc cagggcccca tgctcgcagc 120

ctctgccagg cagtgtcccg acccggatca cgacctgctg tgtcctagct ggaaaccctg 180

gcttcccgcg attgcactcg gggcccacgt catttacagc atttcaatgt gaggtttcta 240

gcagggggag gagtttgcag tggggtgatt ttcaaatgtc ttcacctcac tgcaagagga 300

ggagtttcga acggccgatg tgacatcggc tttttaaccc gtgaagctct gattcccact 360

ccagtccttc gaaagtgtcg ccagggcagg cgacttgatt tgttgtattt gggtctccgg 420

tgaagagctg acgccccctc aaaattggaa acgcatcttc tgaaagatcc tcctgaaatt 480

tctcgatgtt taactgttaa cattttgctg ttgttgtcca cagaaggata acaacagcct 540

ttcaagatcc tccaatagcc taatgccatt gtcctctctg cctcaaaagg aaaacactaa 600

aaatgttggg aacttccgcc actttctata tttgcctttt cctttctagg aattgtgtat 660

agatttttag cttcctttcg ttgtatattg tttttacatt gttattccaa atcacctaat 720

agacactgat caatcaggaa t 741

<210> 20

<211> 346

<212> DNA

＜213〉homo sapiens

<400> 20

cccacgtata ttttttgttg ttttttgttt tttttctgta aaatgtcccg gttcttccat 60

aacttataaa catgatttat accgaggaga tgggaaagtg gacggggcag ggtggactga 120

ccggggatgg ggaagctcct ctcgctgccc cctcggggcg ggcccaggcc ctttggagga 180

tggggacgcc aggacactcc tccctgaggt tgctggccgc ctctgccctg gtgctgtgaa 240

gtcagagccc cgatactccc cgtccacctg ccagttcaca aacttcgact gctgggtctg 300

gatggccatc ttggacctga gaccggggcc cagctggtga atgacc 346

<210> 21

<211> 867

<212> DNA

＜213〉homo sapiens

<400> 21

accatctttt ttttttttct tttatgcgtg aaacttggtg aatctttatt aaactagggt 60

ccaccccagg aggacggctg gggcggggac agggtctccc gctgcaggct gcgcggaggc 120

aggaggcacg gggtggcgtg gggtcgcatg gctgcaggct tcggcgttca gtgattgtcg 180

ctgggcacag gggcggcgct ggtgcccacg gcagcctgca ccttctccac cagcccggcc 240

cactggcgct gcatgtcttc caccaggggc tcgaaccagc tcttgaggcg ggcctggaag 300

gcctcggcct gcaggcgtat ctgctgggcc tgctcctcca gcttggcgcg cacctccgcc 360

acctgctcct tcacctcgtc caggcggtcg cgggtccggc tgcccatctc ctccatccgc 420

gcgcgcagcc gctcgcccca ggcctgggcc cgctcctgta gcggctggcc ggccagggag 480

cccacagtgg cggcccgcac gcggccctgt tccaccaggg gccccaggcg ctcgcggatg 540

gcgctgaggc cgcgctcggc gccctcgcgg gccccggcct ggtacactgc caggcgcttc 600

tgcaggtcat cggcatcgcg gaggagccgc ttacgcagct tgcgcaggtg ggaggcgagg 660

cgcacccgca gctcctcggt gctctggccg agcatggcct gcacctcgcc gcggtactgc 720

accaggcggc cgcacacgtc ctccatgtcc gcgcccagcc gggcctgcgc cgcctgcagc 780

tccttggaca gccgtgcccg cgtctcctcc gccaccgggg tcagttgttc ctccagttcc 840

gatttgtagg ccttcaactc cttcatg 867

<210> 22

<211> 563

<212> DNA

＜213〉homo sapiens

<400> 22

cagcttctct tgcagctcgt gcagcttctg gcgcgcgccc tcttggagct ctgcgcgcag 60

cggctccacc ttctggcggt agagctccat ctcctcctgc cacttcttct ggaagtcgtc 120

cagatccaaa tggcaaacct tcttcatcca ccaggaccca acccacaggc tacttattgc 180

tggaaaccta cgttgttcct tggattgaag taatctctcc ctcttctggt gcgcccacag 240

cacttgcacc aacagtgggt acccaacaga ctagcgtgcc tgccgaagaa ggggtcctct 300

gacaatcagg ggacaatggg gaattatgct ctccagactt tctacacaca caagtcacac 360

aggaaggaag gtaaagagaa actagagaaa ataatttttg aagaaaaaca tttcaggaag 420

tattgaaagt acacggtaac tcagcctggg gcaggggtgg agggcagcag cactgtttgc 480

tgcagctatg ctccttcctc agtgccctgc acacccggga cttgctcggt gagcatctct 540

cgtgtcagtg acagctagtg tga 563

<210> 23

<211> 54

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 23

rururgrura rarargrura rurgrargrc rururgrgru rgrurcrarg rcra 54

<210> 24

<211> 54

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 24

rararurcra rarurgrgrc rurgrururc rurcrurcru rcrurgrgrg rarc 54

<210> 25

<211> 54

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 25

rarcrcrura rurararuru rcrcrargrc rarcrururu rgrargrarg rgrc 54

<210> 26

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 26

cccaccactt gcaactcaga 20

<210> 27

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 27

ttcgggaagt gctcggtttc g 21

<210> 28

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 28

cccagctcag atccatccct t 21

<210> 29

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 29

gcctcttcta tgggtctgtc 20

<210> 30

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 30

gccatgccac ccagagtta 19

<210> 31

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 31

gctgttctct ctctgggact t 21

<210> 32

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 32

gttcccattc ccttattag 19

<210> 33

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 33

cgaatcactt agccactttg 20

<210> 34

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 34

ctgggattac aggcgtgagc 20

<210> 35

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 35

catgtctcct gcctttcctg t 21

<210> 36

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 36

gctcacaata tcaggctgct t 21

<210> 37

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 37

tcaactttac ccagacaagc a 21

<210> 38

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 38

ggacagggta gcaacgccat t 21

<210> 39

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 39

ccacctcagt tgcacggaa 19

<210> 40

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 40

ttgggcatag agtttggttc 20

<210> 41

<211> 54

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 41

rgrurgrgru rcrargrurc rarcrargrc rcrarcrarc rcrargrarc ruru 54

<210> 42

<211> 54

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 42

rgrurcrcrc rururcrurc rcrcrarcrc rarcrarcru rgrurgrarc rurc 54

<210> 43

<211> 54

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 43

rcrargrcra rcrurgrarc rarcrargrc rurarcrcra rcrcrurarc rurc 54

<210> 44

<211> 54

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 44

rurgrurgrc rururcrarc rurgrargrg rarurgrgra rurcrurgrc rcra 54

<210> 45

<211> 54

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 45

rargrargra rararcrgrc rurcrarurc rargrarcra rcrcrarcrc rara 54

<210> 46

<211> 54

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 46

rcrururarg rgrarargrc rarcrararg rargrgrcru rgrargrcrc ruru 54

<210> 47

<211> 54

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 47

rcrcrurgrg rcrurcrcrc rurgrurcrc rcrarcrcru rurgrcrurc rcra 54

<210> 48

<211> 54

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 48

rgrururcra rurururcrc rarcrarcrc rcrargrcru rurcrararc rura 54

<210> 49

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 49

agacctatct gttcccacc 19

<210> 50

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 50

gtcagtcaca gccacaccag 20

<210> 51

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 51

cctatctgtt cccaccttg 19

<210> 52

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 52

gtcacagcca caccagactc t 21

<210> 53

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 53

tcccttctcc caccacactg t 21

<210> 54

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 54

ctcctcaatg tgctcttccc 20

<210> 55

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 55

cccactccca caccagata 19

<210> 56

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 56

aacagcctct tcccagcacc 20

<210> 57

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 57

caccatctac tcctgagccc 20

<210> 58

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 58

tcattgtcac gcgctcccac 20

<210> 59

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 59

tcccagctac tcagaagacc t 21

<210> 60

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 60

tgggcgacag atcaggactc 20

<210> 61

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 61

ctgaggtgca tgggtagtgg t 21

<210> 62

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 62

gccgatcact tgaggtcagg a 21

<210> 63

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 63

attagctggg cgtggtggtg 20

<210> 64

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 64

gctctgcgtg tgggctcatt 20

<210> 65

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 65

gtccctctga tatatgctct c 21

<210> 66

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 66

cttgagttct tcctgcttcc a 21

<210> 67

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 67

tctccagcca gtcacccaga 20

<210> 68

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 68

ccatcaacat ctccctcagc c 21

<210> 69

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 69

cttcttcctc tttccctctt 20

<210> 70

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 70

acagcagcac tcaggaggca 20

<210> 71

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 71

gggtagttct gagggcacga 20

<210> 72

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 72

aggcaggtgg atgtcagtg 19

<210> 73

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 73

gggtagggtt cagagaaggc 20

<210> 74

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 74

actcctcctc ttgcagtgag 20

<210> 75

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 75

gctgttgtta tccttctgtg 20

<210> 76

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 76

atcgcgggaa gccagggttt 20

<210> 77

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 77

gccagaatag gttgagaggg a 21

<210> 78

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 78

gccgttcgaa actcctcctc t 21

<210> 79

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 79

ggtttccgct gtgctctgtg 20

<210> 80

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 80

acgttgcctc agaatctccc 20

<210> 81

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 81

ttcctcaatc aacctcttcc t 21

<210> 82

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 82

gtgatgtttc ctctgttgtc 20

<210> 83

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 83

cactttctag ctgcaccacc 20

<210> 84

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 84

ccgtccactt tcccatctcc t 21

<210> 85

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 85

cccgtccact ttcccatctc 20

<210> 86

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 86

cttcacagca ccagggcaga 20

<210> 87

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 87

cccagcagtc gaagtttgtg a 21

<210> 88

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 88

ccggtctcag gtccaagatg 20

<210> 89

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 89

tggtggagaa ggtgcaggct 20

<210> 90

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 90

gattcaccaa gtttcacgca t 21

<210> 91

<211> 20

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 91

gacgaggtga aggagcaggt 20

<210> 92

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 92

tcggaactgg aggaacaact g 21

<210> 93

<211> 13

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 93

tggagctcag ttt 13

<210> 94

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 94

tttctcgtgc agct 14

<210> 95

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 95

ttctggcgcg tgcc 14

<210> 96

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 96

ccctcgtgga gctc 14

<210> 97

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 97

gcccgcgcgc agcg 14

<210> 98

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 98

cggctccacc ttct 14

<210> 99

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 99

ctggcggtag agct 14

<210> 100

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 100

gctcgtgcag cttc 14

<210> 101

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 101

cagcttctgg cgcg 14

<210> 102

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 102

gcgcgcagcg gctc 14

<210> 103

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 103

tccaccttct ggcg 14

<210> 104

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 104

cggtgtagag ctcc 14

<210> 105

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 105

ccatctcctc ctgc 14

<210> 106

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 106

gaagtcgtcc agatccaaa 19

<210> 107

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 107

aagtcgtcca gatccaaat 19

<210> 108

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 108

agtcgtccag atccaaatg 19

<210> 109

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 109

gtcgtccaga tccaaatgg 19

<210> 110

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 110

tcgtccagat ccaaatggc 19

<210> 111

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 111

cgtccagatc caaatggca 19

<210> 112

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 112

gtccagatcc aaatggcaa 19

<210> 113

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 113

tccagatcca aatggcaaa 19

<210> 114

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 114

cagatccaaa tg 12

<210> 115

<211> 16

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 115

agatccaaat ggcaaa 16

<210> 116

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 116

agatccaaat gg 12

<210> 117

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 117

gatccaaatg gc 12

<210> 118

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 118

atccaaatgg ca 12

<210> 119

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 119

tccaaatggc aa 12

<210> 120

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 120

ccaaatggca aa 12

<210> 121

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 121

ccaaatggca aaccttctt 19

<210> 122

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 122

atggcaaatc ttcttcatc 19

<210> 123

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 123

aaatggcaaa ccttcttca 19

<210> 124

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 124

aaatggcaaa tcttcttca 19

<210> 125

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 125

atggcaaacc ttcttcatc 19

<210> 126

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 126

ccaaatggca aatcttctt 19

<210> 127

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 127

atggcaaacc ttctt 15

<210> 128

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 128

atggcaaatc ttctt 15

<210> 129

<211> 11

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 129

cttcttcatc c 11

<210> 130

<211> 17

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 130

ccaggaccca acccaca 17

<210> 131

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 131

gctacttatt gctg 14

<210> 132

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 132

tgctggaaac ctac 14

<210> 133

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 133

ttccttggat tgaa 14

<210> 134

<211> 16

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 134

gcccacagca cttgca 16

<210> 135

<211> 16

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 135

caccaacagt gggtac 16

<210> 136

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 136

caacagacta gc 12

<210> 137

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 137

gaagaagggg tcct 14

<210> 138

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 138

gaattatgct ctcc 14

<210> 139

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 139

ctccagactt tcta 14

<210> 140

<211> 16

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 140

aagtcacaca ggaagg 16

<210> 141

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 141

gaaactagag aaaa 14

<210> 142

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 142

aataattttt gaag 14

<210> 143

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 143

gaagtattga aagt 14

<210> 144

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 144

tgaaagtaca cggt 14

<210> 145

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 145

ctggggcagg ggtg 14

<210> 146

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 146

ggggtggagg gcag 14

<210> 147

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 147

tgctccttcc tcag 14

<210> 148

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 148

acccgggact tgctc 15

<210> 149

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 149

tgtcagtgac agct 14

<210> 150

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 150

tgtcagtgac agctagtgt 19

<210> 151

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 151

gtcagtgaca gctagtgtg 19

<210> 152

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 152

tcagtgacag ctagtgtga 19

<210> 153

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 153

agtgacagct agtgtgagt 19

<210> 154

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 154

tgacagctag tgtga 15

<210> 155

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 155

tgacagctag tgtgagtac 19

<210> 156

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 156

gacagctagt gtgagtact 19

<210> 157

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 157

cagctagtgt gagtactct 19

<210> 158

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 158

agctagtgtg agtactctt 19

<210> 159

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 159

gctagtgtga gtactctta 19

<210> 160

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 160

ctagtgtgag tactcttat 19

<210> 161

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 161

ctagtgtgag tact 14

<210> 162

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 162

tagtgtgagt actcttatg 19

<210> 163

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 163

agtgtgagta ctcttatgt 19

<210> 164

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 164

gtgtgagtac tcttatgtt 19

<210> 165

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 165

tgtgagtact cttatgttc 19

<210> 166

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 166

gtgagtactc ttatgttca 19

<210> 167

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 167

tgagtactct tatgttcag 19

<210> 168

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 168

actcttatgt tcag 14

<210> 169

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 169

tacctcttga cttt 14

<210> 170

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 170

gactttgggg acaa 14

<210> 171

<211> 13

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 171

aaactgagct cca 13

<210> 172

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 172

agctgcacga gaaa 14

<210> 173

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 173

ggcacgcgcc agaa 14

<210> 174

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 174

gagctccacg aggg 14

<210> 175

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 175

cgctgcgcgc gggc 14

<210> 176

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 176

agaaggtgga gccg 14

<210> 177

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 177

agctctaccg ccag 14

<210> 178

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 178

gaagctgcac gagc 14

<210> 179

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 179

cgcgccagaa gctg 14

<210> 180

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 180

gagccgctgc gcgc 14

<210> 181

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 181

cgccagaagg tgga 14

<210> 182

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 182

ggagctctac accg 14

<210> 183

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 183

gcaggaggag atgg 14

<210> 184

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 184

tttggatctg gacgacttc 19

<210> 185

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 185

atttggatct ggacgactt 19

<210> 186

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 186

catttggatc tggacgact 19

<210> 187

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 187

ccatttggat ctggacgac 19

<210> 188

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 188

gccatttgga tctggacga 19

<210> 189

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 189

tgccatttgg atctggacg 19

<210> 190

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 190

ttgccatttg gatctggac 19

<210> 191

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 191

tttgccattt ggatctgga 19

<210> 192

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 192

catttggatc tg 12

<210> 193

<211> 16

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 193

tttgccattt ggatct 16

<210> 194

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 194

ccatttggat ct 12

<210> 195

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 195

gccatttgga tc 12

<210> 196

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 196

tgccatttgg at 12

<210> 197

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 197

tgccatttgg at 12

<210> 198

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 198

tttgccattt gg 12

<210> 199

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 199

aagaaggttt gccatttgg 19

<210> 200

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 200

gatgaagaag atttgccat 19

<210> 201

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 201

tgaagaaggt ttgccattt 19

<210> 202

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 202

tgaagaagat ttgccattt 19

<210> 203

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 203

gatgaagaag gtttgccat 19

<210> 204

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 204

aagaagattt gccatttgg 19

<210> 205

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 205

aagaaggttt gccat 15

<210> 206

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 206

aagaagattt gccat 15

<210> 207

<211> 11

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 207

ggatgaagaa g 11

<210> 208

<211> 17

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 208

tgtgggttgg gtcctgg 17

<210> 209

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 209

cagcaataag tagc 14

<210> 210

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 210

gtaggtttcc agca 14

<210> 211

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 211

ttcaatccaa ggaa 14

<210> 212

<211> 16

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 212

tgcaagtgct gtgggc 16

<210> 213

<211> 16

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 213

gtacccactg ttggtg 16

<210> 214

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 214

gctagtctgt tg 12

<210> 215

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 215

aggacccctt cttc 14

<210> 216

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 216

ggagagcata attc 14

<210> 217

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 217

tagaaagtct ggag 14

<210> 218

<211> 16

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 218

ccttcctgtg tgactt 16

<210> 219

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 219

ttttctctag tttc 14

<210> 220

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 220

cttcaaaaat tatt 14

<210> 221

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 221

actttcaata cttc 14

<210> 222

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 222

accgtgtact ttca 14

<210> 223

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 223

cacccctgcc ccag 14

<210> 224

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 224

ctgccctcca cccc 14

<210> 225

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 225

ctgaggaagg agca 14

<210> 226

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 226

gagcaagtcc cgggt 15

<210> 227

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 227

agctgtcact gaca 14

<210> 228

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 228

acactagctg tcactgaca 19

<210> 229

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 229

cacactagct gtcactgac 19

<210> 230

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 230

tcacactagc tgtcactga 19

<210> 231

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 231

actcacacta gctgtcact 19

<210> 232

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 232

tcacactagc tgtca 15

<210> 233

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 233

gtactcacac tagctgtca 19

<210> 234

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 234

agtactcaca ctagctgtc 19

<210> 235

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 235

agagtactca cactagctg 19

<210> 236

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 236

aagagtactc acactagct 19

<210> 237

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 237

taagagtact cacactagc 19

<210> 238

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 238

ataagagtac tcacactag 19

<210> 239

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 239

agtactcaca ctag 14

<210> 240

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 240

cataagagta ctcacacta 19

<210> 241

<211> 19

<212> DNA

<213> Artificial Seqiuence

<220>

＜223〉antisense oligonucleotide

<400> 241

acataagagt actcacact 19

<210> 242

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 242

aacataagag tactcacac 19

<210> 243

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 243

gaacataaga gtactcaca 19

<210> 244

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 244

tgaacataag agtactcac 19

<210> 245

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 245

ctgaacataa gagtactca 19

<210> 246

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 246

ctgaacataa gagt 14

<210> 247

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 247

aaagtcaaga ggta 14

<210> 248

<211> 14

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 248

ttgtccccaa agtc 14

<210> 249

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 249

aagaaggttt gccat 15

<210> 250

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 250

tcacactagc tgtca 15

<210> 251

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 251

aagaaggttt gccat 15

<210> 252

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 252

tcacactagc tgtca 15

<210> 253

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 253

ctcctcctgc cacttcttct g 21

<210> 254

<211> 22

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 254

ctggtggatg aagaaggttt gc 22

<210> 255

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 255

tttggatctg gacgacttc 19

<210> 256

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 256

caataagtag cctgt 15

<210> 257

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 257

cacgctagtc tgttg 15

<210> 258

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 258

cttccttcct gtgtg 15

<210> 259

<211> 15

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 259

caggctgagt taccg 15

<210> 260

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 260

gctagtctgt tg 12

<210> 261

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 261

gtctgatgga ga 12

<210> 262

<211> 6

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 262

gctagt 6

<210> 263

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 263

tgccatttgg atctggacg 19

<210> 264

<211> 48

<212> DNA

＜213〉artificial sequence

<220>

＜223〉the reverse complemental body of antisense oligonucleotide SEQ ID NO:23

<400> 264

rcrurgrarc rarcrcrara rgrcrurcra rurarcruru rurarcaa 48

<210> 265

<211> 48

<212> DNA

＜213〉artificial sequence

<220>

＜223〉the reverse complemental body of antisense oligonucleotide SEQ ID NO:24

<400> 265

rcrcrcrarg rargrargra rgrararcra rgrcrcraru rurgratt 48

<210> 266

<211> 48

<212> DNA

＜213〉artificial sequence

<220>

＜223〉the reverse complemental body of antisense oligonucleotide SEQ ID NO:25

<400> 266

rcrurcrurc rararargru rgrcrurgrg rararurura rurarggt 48

<210> 267

<211> 48

<212> DNA

＜213〉artificial sequence

<220>

＜223〉the reverse complemental body of antisense oligonucleotide SEQ ID NO:41

<400> 267

rgrurcrurg rgrurgrurg rgrcrurgru rgrarcrurg rarcrcac 48

<210> 268

<211> 48

<212> DNA

＜213〉artificial sequence

<220>

＜223〉the reverse complemental body of antisense oligonucleotide SEQ ID NO:42

<400> 268

rgrurcrarc rargrurgru rgrgrurgrg rgrargrara rgrgrgac 48

<210> 269

<211> 48

<212> DNA

＜213〉artificial sequence

<220>

＜223〉the reverse complemental body of antisense oligonucleotide SEQ ID NO:43

<400> 269

rgrurargrg rurgrgrura rgrcrurgru rgrurcrarg rurgrctg 48

<210> 270

<211> 48

<212> DNA

＜213〉artificial sequence

<220>

＜223〉the reverse complemental body of antisense oligonucleotide SEQ ID NO:44

<400> 270

rgrcrargra rurcrcraru rcrcrurcra rgrurgrara rgrcraca 48

<210> 271

<211> 48

<212> DNA

＜213〉artificial sequence

<220>

＜223〉the reverse complemental body of antisense oligonucleotide SEQ ID NO:45

<400> 271

rgrgrurgrg rurgrurcru rgrarurgra rgrcrgruru rurcruct 48

<210> 272

<211> 48

<212> DNA

＜213〉artificial sequence

<220>

＜223〉the reverse complemental body of antisense oligonucleotide SEQ ID NO:46

<400> 272

rgrgrcrurc rargrcrcru rcrururgru rgrcrururc rcruraag 48

<210> 273

<211> 48

<212> DNA

＜213〉artificial sequence

<220>

＜223〉the reverse complemental body of antisense oligonucleotide SEQ ID NO:47

<400> 273

rgrargrcra rargrgrurg rgrgrarcra rgrgrgrarg rcrcragg 48

<210> 274

<211> 48

<212> DNA

＜213〉artificial sequence

<220>

＜223〉the reverse complemental body of antisense oligonucleotide SEQ ID NO:48

<400> 274

rgrururgra rargrcrurg rgrgrurgru rgrgrarara rurgraac 48

<210> 275

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉probe sequence

<400> 275

tttggatctg gacgacttc 19

<210> 276

<211> 21

<212> DNA

＜213〉artificial sequence

<220>

＜223〉forward primer

<400> 276

ctcctcctgc cacttcttct g 21

<210> 277

<211> 22

<212> DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer

<400> 277

ctggtggatg aagaaggttt gc 22

<210> 278

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 278

gctagtctgt tg 12

<210> 279

<211> 12

<212> DNA

＜213〉artificial sequence

<220>

＜223〉antisense oligonucleotide

<400> 279

gtctgatgga ga 12

Claims (9)

1. at least a antisense oligonucleotide in the zone of the natural antisense oligonucleotide of targeting lipid transfer and metabolic gene polynucleotide is in preparation is used for body or the application in the medicine of the function of external adjusting patient's cell or tissue lipid transfer and metabolic gene polynucleotide and/or expression.

2. the application of claim 1, wherein with respect to contrast, the function of described lipid transfer and metabolic gene and/or express in vivo or external increase.

3. the application of claim 1, the natural antisense sequence of wherein said at least a antisense oligonucleotide targeting lipid transfer and metabolic gene polynucleotide.

4. the application of claim 1, wherein said at least a antisense oligonucleotide targeting comprises the coding of lipid transfer and metabolic gene polynucleotide and/or the nucleotide sequence of non-coding nucleic acid sequence.

5. the application of claim 1, the overlapping and/or non-overlapping sequence of wherein said at least a antisense oligonucleotide targeting lipid transfer and metabolic gene polynucleotide.

6. the application of claim 1, wherein said at least a antisense oligonucleotide comprises one or more modifications, described modification is selected from: the nucleotide of bonding, at least a modification and its combination between the sugar moieties of at least a modification, the nucleoside of at least a modification.

7. the application of claim 6, wherein said one or more modifications comprise the sugar moieties of at least a modification, and the sugar moieties of described at least a modification is selected from the sugar moieties of 2'-O-methoxy ethyl modification, the sugar moieties that the 2'-methoxyl group is modified, sugar moieties, dicyclo sugar moieties and the combination thereof that the 2'-O-alkyl is modified.

8. the application of claim 6, wherein said one or more modifications comprise bonding between the nucleoside of at least a modification, and bonding is selected from thiophosphate, 2'-O methoxy ethyl (MOE), 2'-fluorine, phosphonate ester, phosphorodithioate, alkyl thio-phosphonate, phosphoramidate, carbamate, carbonic ester, phosphotriester, imidic acid ethyl ester, carboxymethyl ester and its combination between the nucleoside of described at least a modification.

9. the application of claim 6, wherein said one or more modifications comprise the nucleotide of at least a modification, the nucleotide of described at least a modification is selected from peptide nucleic acid(PNA) (PNA), lock nucleic acid (LNA), arabinonucleic acid (FANA), its analog, derivant and combination.

Images